JP5968458B2 - 幹細胞を若くするための培地組成物 - Google Patents
幹細胞を若くするための培地組成物 Download PDFInfo
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Description
前記間葉系幹細胞の培養に用いられる基本培地としては、当業界で幹細胞培養に適していると知られている通常の培地、例えばDMEM、MEM、K−SFM培地などが用いられるが、好ましくは、無血清培地が用いられ、最も好ましくはK−SFM(Keratinocyte-SFM;Keratinocyte serum free medium)培地が用いられる。
・幹細胞因子(SCF、Steel因子)、c−kitを二量化する他のリガンドまたは抗体、および同じシグナル伝達経路の他の活性剤
・他のチロシンキナーゼ関連受容体、例えば、血小板由来の成長因子(Platelet-Derived Growth Factor;PDGF)、マクロファージコロニー刺激因子、Flt−3リガンド、および血管内皮増殖因子(Vascular Endothelial Growth Factor;VEGF)の受容体のためのリガンド
・環状AMP濃度を高める因子、例えば、フォルスコリン
・gp130を誘導する因子、例えばLIFまたはオンコスタチンM
・造血成長因子、例えば、トロンボポエチン(TPO)
・変形性成長因子、例えば、TGFβ1
・ニューロトロフィン、例えば、CNTF
患者の年齢が20代、30代、70代と80代の患者からの脂肪吸引法(Liposuction)によって、腹部の脂肪から脂肪組織を各々分離し、PBSで洗浄した。洗浄された脂肪組織を細かく切った後、コラゲナーゼタイプ1(1mg/mL)を添加したDMEM培地を用いて、37℃で2時間の間組織を分解させた。コラゲナーゼ処理された組織をPBSで洗浄した後、1000rpmで5分間遠心分離し、上澄み液を除去し、ペレットをPBSで洗浄した後、1000rpmで5分間遠心分離した。100μmメッシュでフィルタリングして浮遊物を除去した後、PBSで洗浄し、10% FBS、2mM NAC(N−アセチル−L−システイン)、0.2mMアスコルビン酸が添加されたDMEM培地で培養した。
その結果、20代、40代と70代で分離された脂肪幹細胞が3継代培養後、それぞれ類似する幹細胞の活性を有することが確認された。
実施例1で用いられたRKCM培地に添加される活性成分であるFBS、NAC、アスコルビン酸、カルシウム、rEGF、5μg/mLのインスリン、bFGF、ヒドロコルチゾンおよびセレン(selenium)のうちひとつずつを除去した培地を製造し、前記活性成分がそれぞれ除去された培地での20代、30代、70代、及び80代の患者由来の脂肪幹細胞の表現型を確認した。
実施例1の方法で分離された20代、30代、70代、及び80代由来の脂肪幹細胞を前記各々の培地で第4代まで継代した後、顕微鏡像での形態を確認し(図1及び図2)、継代の過程で細胞の数を血球計算器を用いて測定し、細胞数をカウントして増殖速度を確認した。
実施例1の方法で分離された20代、30代、70代、及び80代由来の脂肪幹細胞を、前記5種(1、2、3、9、10)培地で第3代まで継代した後、幹細胞のテロメラーゼ活性を確認した。
その結果、図7に示すように、全年齢層と二つの培地ですべてP6よりも継代数の低いP4の活性度が高く示されるとの結果を確認することができた。
実施例1の方法で分離された20代、30代、70代、及び80代由来の脂肪幹細胞を、前記1、9番培地で第3代まで継代した後、90%まで育てた。このように育てた脂肪幹細胞から培地を除去し、PBSで1回以上洗浄した後、RNA抽出のための細胞溶解バッファー(Intron Biotechnolgy、Sungnam、Korea)を入れて細胞を溶解させて、Total Extractionキット(Intron Biotechnolgy)を利用して、RNAを抽出した。抽出されたRNAをcDNA(Intron Biotechnology cDNA syntheis kit)に変換した後、多能性マーカーであるoct−4、NanogおよびRex1のプライマーを作成してからPCRを実施した。PCR産物を電気泳動した後、イメージアナライザーを通じて定量した。
実施例1の方法で分離された20代、30代、70代、及び80代由来の脂肪幹細胞を5種(1、2、3、9、10)培地で第3代まで継代した後、脂肪誘導培地であるNH Adipodiff培地(Miltenyi Biotec、Bergisch Gladbach、Germany)で37℃、5%のCO2で21日間培養を進めて、脂肪細胞分化培地の交換は2日間隔で行われた。
Claims (3)
- FBS(fetal bovine serum)、セレン、サイトカインとしてのEGFまたはbFGF、およびNAC(N−アセチル−L−システイン)を含有することを特徴とする培地で、60〜120歳の高齢者由来の間葉系幹細胞を培養することを特徴とする、60〜120歳の高齢者由来の間葉系幹細胞を20〜30代の若者由来の幹細胞様に転換する方法。
- 前記培地は、インスリンまたはインスリン様因子を追加で含有することを特徴とする請求項1に記載の方法。
- 前記培地は、ヒドロコルチゾンを追加で含有することを特徴とする請求項1または2に記載の方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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KR10-2011-0127885 | 2011-12-01 | ||
KR20110127885 | 2011-12-01 | ||
PCT/KR2012/010380 WO2013081436A1 (ko) | 2011-12-01 | 2012-12-03 | 줄기세포를 젊게 만들기 위한 배지 조성물 |
Publications (2)
Publication Number | Publication Date |
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JP2015500018A JP2015500018A (ja) | 2015-01-05 |
JP5968458B2 true JP5968458B2 (ja) | 2016-08-10 |
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Application Number | Title | Priority Date | Filing Date |
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JP2014544678A Active JP5968458B2 (ja) | 2011-12-01 | 2012-12-03 | 幹細胞を若くするための培地組成物 |
Country Status (11)
Country | Link |
---|---|
US (1) | US9708584B2 (ja) |
EP (1) | EP2811015B1 (ja) |
JP (1) | JP5968458B2 (ja) |
KR (2) | KR20130061658A (ja) |
CN (1) | CN104114691B (ja) |
AU (1) | AU2012346645B2 (ja) |
BR (1) | BR112014012972A2 (ja) |
CA (1) | CA2856764C (ja) |
ES (1) | ES2827207T3 (ja) |
PH (1) | PH12014501175A1 (ja) |
WO (1) | WO2013081436A1 (ja) |
Families Citing this family (17)
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EP2840133B1 (en) * | 2012-04-18 | 2017-06-14 | Jeong Chan Ra | Method for manufacturing stem cell having appropriate size for intravascular administration |
KR101523040B1 (ko) * | 2012-06-26 | 2015-05-26 | 라정찬 | 고농도의 줄기세포 제조방법 |
AU2015243236B2 (en) | 2014-04-11 | 2020-12-03 | The Administrators Of The Tulane Educational Fund | Cell-surface marker of early MSC aging |
KR20160145778A (ko) * | 2014-07-07 | 2016-12-20 | 메디포스트(주) | 작은 크기 줄기세포의 발모 촉진능 및 이의 용도 |
CA2958533C (en) * | 2014-08-21 | 2023-03-14 | Ajinomoto Co., Inc. | Culture medium for mesenchymal stem cells |
KR102048067B1 (ko) * | 2015-01-19 | 2019-11-22 | 서울대학교병원 | 인간 중간엽 줄기세포의 줄기세포능을 증가시키는 방법 |
CN104611295A (zh) * | 2015-02-06 | 2015-05-13 | 华南农业大学 | 一种食蟹猴睾丸间质祖细胞永生系的建立方法 |
EP3292198A4 (en) * | 2015-05-05 | 2018-10-17 | The J. David Gladstone Institutes, A Testamentary Trust Established under The Will of J. David Gladstone | Reversion of primed pluripotent stem cells to naive pluripotent stem cells |
US11984312B2 (en) * | 2016-03-30 | 2024-05-14 | University of Pittsburgh—of the Commonwealth System of Higher Education | System for portable gas storage and delivery |
US10894947B1 (en) * | 2016-04-29 | 2021-01-19 | Hope Biosciences, Llc | Method for generating protein rich conditioned medium |
JP7076709B2 (ja) * | 2016-08-18 | 2022-05-30 | 北海道公立大学法人 札幌医科大学 | 間葉系幹細胞活性化剤 |
KR102100608B1 (ko) | 2017-06-30 | 2020-04-14 | 주식회사 차바이오텍 | 줄기세포 유래 피부전구세포 배양액 및 이의 제조 방법 |
CN113801836A (zh) * | 2020-06-16 | 2021-12-17 | 台湾粒线体应用技术股份有限公司 | 提升线粒体功能的培养方法及其使用的组合物 |
CN112881113B (zh) * | 2021-01-12 | 2023-06-16 | 武汉呵尔医疗科技发展有限公司 | 一种稳定的粘液处理剂 |
CN113583937B (zh) * | 2021-07-02 | 2023-02-10 | 深圳市北科生物科技有限公司 | 用于哺乳动物多能干细胞分化成定型内胚层细胞的培养基和培养方法 |
WO2023200363A1 (ru) * | 2022-04-13 | 2023-10-19 | Фания Иршатовна МАГАНОВА | Способ получения пероральной формы субстанции, содержащей инсулин |
CN115747151B (zh) * | 2022-11-25 | 2024-06-21 | 南开大学 | 一种通过调控端粒以稳定扩增脂肪干细胞的方法 |
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EP2348104A1 (en) | 1999-08-05 | 2011-07-27 | Mcl Llc | Multipotent adult stem cells and methods for isolation |
US8147824B2 (en) * | 1999-08-05 | 2012-04-03 | Athersys, Inc. | Immunomodulatory properties of multipotent adult progenitor cells and uses thereof |
FR2814752A1 (fr) * | 2000-10-02 | 2002-04-05 | Chru Lille | Procede d'obtention in vitro de cellules insulino- secretrices de mammifere et leurs utilisations |
KR100679642B1 (ko) | 2005-11-16 | 2007-02-06 | 주식회사 알앤엘바이오 | 인간 지방조직 유래 다분화능 줄기세포 및 이를 함유하는세포치료제 |
JP2009539546A (ja) | 2006-06-15 | 2009-11-19 | アールエヌエル バイオ カンパニー,リミティッド | ヒト脂肪組織由来成体幹細胞、繊維芽細胞、及び脂肪又は脂肪細胞を含有する皮膚美容又は形成用の組成物 |
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KR20100008763A (ko) * | 2008-07-16 | 2010-01-26 | 주식회사 알앤엘바이오 | 지방조직 유래 다분화능 줄기세포의 배양물 및 그 추출 단백질을 함유한 화장료용 조성물 |
EP2192174B1 (en) * | 2008-11-21 | 2015-11-11 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Reprogramming cells toward a pluripotent state |
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EP2511365A4 (en) * | 2009-10-23 | 2013-07-03 | Rnl Bio Co Ltd | METHOD FOR INDUCING MIGRATION OF ADULT STEM CELLS DERIVED FROM ADIPOSE TISSUE |
CN101735980B (zh) | 2010-02-11 | 2011-07-27 | 中国人民解放军总医院 | 体外培养骨髓间充质干细胞的完全培养基 |
KR20120057784A (ko) | 2010-11-29 | 2012-06-07 | 주식회사 알앤엘바이오 | 줄기세포의 안정성 증진용 조성물 |
US20120276064A1 (en) * | 2011-04-05 | 2012-11-01 | Blau Helen M | Methods and compositions for rejuvenation and expansion of stem cells |
EP2840133B1 (en) * | 2012-04-18 | 2017-06-14 | Jeong Chan Ra | Method for manufacturing stem cell having appropriate size for intravascular administration |
KR101523040B1 (ko) * | 2012-06-26 | 2015-05-26 | 라정찬 | 고농도의 줄기세포 제조방법 |
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- 2012-12-03 CA CA2856764A patent/CA2856764C/en active Active
- 2012-12-03 US US14/360,621 patent/US9708584B2/en active Active
- 2012-12-03 KR KR1020120138985A patent/KR20130061658A/ko active Search and Examination
- 2012-12-03 WO PCT/KR2012/010380 patent/WO2013081436A1/ko active Application Filing
- 2012-12-03 AU AU2012346645A patent/AU2012346645B2/en active Active
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Also Published As
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AU2012346645B2 (en) | 2015-08-27 |
BR112014012972A2 (pt) | 2017-06-13 |
CN104114691B (zh) | 2017-05-03 |
EP2811015A1 (en) | 2014-12-10 |
CA2856764A1 (en) | 2013-06-06 |
US20140322811A1 (en) | 2014-10-30 |
KR20130061658A (ko) | 2013-06-11 |
JP2015500018A (ja) | 2015-01-05 |
EP2811015A4 (en) | 2015-01-21 |
KR101598813B1 (ko) | 2016-03-02 |
CA2856764C (en) | 2018-04-03 |
KR20150093645A (ko) | 2015-08-18 |
PH12014501175B1 (en) | 2014-10-20 |
US9708584B2 (en) | 2017-07-18 |
WO2013081436A1 (ko) | 2013-06-06 |
EP2811015B1 (en) | 2020-09-02 |
CN104114691A (zh) | 2014-10-22 |
ES2827207T3 (es) | 2021-05-20 |
AU2012346645A1 (en) | 2014-06-26 |
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