JP6825182B2 - 薬剤前処理による間葉系幹細胞由来エクソソームの調製方法 - Google Patents
薬剤前処理による間葉系幹細胞由来エクソソームの調製方法 Download PDFInfo
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Description
本発明は、効率的な間葉系幹細胞由来エクソソームの調製方法を提供することを目的とするものである。
間葉系幹細胞の培地にスタチン系薬剤を加え、12〜24時間前処理した後、細胞の培地を無エクソソームの完全培地に変更して引き続き培養し、48時間後に馴化培地を収集し、スタチン系薬剤で前処理した間葉系幹細胞から分泌されたエクソソームを超遠心により分離して得ることを含む。
馴化培地を収集した後、遠心により細胞を除去する工程と、遠心により細胞破片を除去する工程と、高速遠心により大型小胞を除去する工程と、そして超遠心により沈殿を回収し、再懸濁後に再度超遠心し、エクソソームを得る工程とをこの順で含む。
間葉系幹細胞由来エクソソームの調製方法
差次接着により初代ラット(SDラット、60〜80g)BM−MSCsを単離し、3〜4代目に拡大継代して使用に備えた。BM−MSCs完全培地(IMDM、Invitrogen、アメリカ)にATVを加え、24時間前処理した後、細胞の培地を無エクソソームの完全培地(18時間の超遠心により得られた10%FBS含有IMDM)に変更して引き続き培養した。48時間後、馴化培地を回収し、超遠心によりATVで前処理したBM−MSCsに分泌されたエクソソーム(MSCATV−Exo)を分離して得た。具体的な超遠心工程は、馴化培地を回収した後、300gで10分遠心することにより、細胞を除去する工程と、2000gで20分遠心することにより、細胞破片を除去する工程と、16500gで30分高速遠心することにより、大型小胞を除去する工程と、120000gで70分超遠心することにより沈殿を回収し、再懸濁後に、再度120000gで70分超遠心し、エクソソームを得る工程とを含む。
電子顕微鏡(HITACHI、H−600IV、日本)で、形態構造を解析観察すること、NTA(Malvern Instruments、NanoSight、イギリス)で、エクソソームの粒径分布を分析すること、及びWestern Blotでエクソソーム蛋白質マーカーを検出することを含む。
超遠心により分離して得たMSCATV−Exoは、電子顕微鏡下で球状若しくはディスク状を示し、100nm程度の大きさを有する。NTA分析により、その粒径分布は30〜150nmの範囲にあり、Western Blotアッセイにより、MSCATV−ExoはTSG101、Alix、CD63、CD81などのエクソソーム蛋白質マーカーを高発現していることが明らかとなる。ATVで前処理されたBM−MSCと前処理されていないBM−MSCは、それぞれの分泌されたエクソソームが、形態、粒径分布及び蛋白質マーカーに有意な差が認められていない。具体的な結果として、図1A〜図1Cに示した。なお、図1Aは、間葉系幹細胞由来エクソソーム(MSC−Exo)の形態構造が電子顕微鏡下で観察されたものであり、球状若しくはディスク状を示し、大きさが100nm程度であり、スタチン系前処理した後、形態が変化しなっかた。図1Bでは、MSC−Exoの粒径分布がNTAにより分析され、スタチン系前処理されたと処理されていないMSC−Exoの粒径分布は、ともに30〜150nmの範囲にある。図1Cでは、エクソソームの蛋白質マーカー同定を示し、スタチン系処理したMSC−Exoは、TSG101、Alix、CD63、CD81などのエクソソーム蛋白質マーカーを高発現している。
Claims (3)
- 間葉系幹細胞の培地に1μMのアトルバスタチンを加え、24時間前処理した後、細胞の培地を無エクソソームの完全培地に変更して引き続き培養し、48時間後に馴化培地を収集し、アトルバスタチンで前処理した間葉系幹細胞から分泌されたエクソソームを超遠心により分離して得ることを含み、前記アトルバスタチンで前処理した間葉系幹細胞から得られるエクソソームは、球状又はディスク状であり、30〜150nmの範囲の粒径分布を有し、かつ、アトルバスタチンで前処理していない間葉系幹細胞から得られるエクソソームよりも10倍以上のlncRNA H19を高発現している、間葉系幹細胞由来エクソソームの調製方法。
- 前記超遠心は、
馴化培地を収集し、300gで10分遠心することにより、細胞を除去する工程と、
2000gで20分遠心することにより、細胞破片を除去する工程と、
16500gで30分高速遠心することにより、大型小胞を除去する工程と、
120000gで70分超遠心することにより沈殿を回収し、再懸濁後に、再度120000gで70分超遠心し、エクソソームを得る工程とを含む、請求項1に記載の方法。 - 前記間葉系幹細胞は、骨髄間葉系幹細胞又は脂肪間葉系幹細胞を含む、請求項1に記載の方法。
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KR102184428B1 (ko) * | 2020-05-25 | 2020-11-30 | 주식회사 씨케이엑소젠 | 중배엽줄기세포 유래의 엑소좀 생산방법 및 이로부터 제조된 배양액 |
CN111944747A (zh) * | 2020-08-14 | 2020-11-17 | 福建医科大学附属协和医院 | 一种用于治疗心肌梗死的人脂肪间充质干细胞外泌体及其用途 |
CN111925983A (zh) * | 2020-08-14 | 2020-11-13 | 福建医科大学附属协和医院 | 一种用于治疗心肌梗死的高表达il-10的人脂肪间充质干细胞外泌体的制备方法 |
CN112359013A (zh) * | 2020-11-17 | 2021-02-12 | 广州赛莱拉干细胞科技股份有限公司 | 一种间充质干细胞外泌体的制备方法和应用 |
JP2023148677A (ja) | 2022-03-30 | 2023-10-13 | 株式会社山田養蜂場本社 | エクソソーム分泌促進剤 |
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US9072816B2 (en) * | 2006-01-18 | 2015-07-07 | Cormatrix Cardiovascular, Inc. | Composition for modulating inflammation of cardiovascular tissue |
CN101129356A (zh) * | 2007-08-01 | 2008-02-27 | 中国医学科学院阜外心血管病医院 | 洛伐他汀在制备抑制骨髓间充质干细胞凋亡药物上的应用 |
US20140341882A1 (en) * | 2011-09-13 | 2014-11-20 | Takahiro Ochiya | Pharmaceutical product for preventing or treating alzheimer's disease |
AU2015330855A1 (en) * | 2014-10-09 | 2017-04-27 | Celularity Inc. | Placenta-derived adherent cell exosomes and uses thereof |
JP6110578B2 (ja) * | 2014-11-10 | 2017-04-05 | 学校法人大阪医科薬科大学 | 幹細胞機能増強用スタチン封入ナノ粒子製剤、並びにそれを含有する機能増強幹細胞及びその製造方法 |
TWI601741B (zh) * | 2016-07-11 | 2017-10-11 | 財團法人國家衛生研究院 | 利用前列腺素受體ep4-拮抗劑誘導幹細胞製造含有高囊泡含物之外泌體囊泡的方法及其應用 |
CN106282107A (zh) * | 2016-08-30 | 2017-01-04 | 章毅 | 人胎盘间充质干细胞源分离外泌体的方法及其应用 |
CN107137700B (zh) * | 2017-04-27 | 2021-01-12 | 张国瑜 | 一种基于干细胞源外泌体的组合物及其在制备治疗心肌梗死的药物中的应用 |
CN108728410B (zh) * | 2018-06-19 | 2020-04-21 | 中国医学科学院阜外医院 | 基于药物预处理的间充质干细胞来源外泌体的制备方法 |
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2018
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- 2018-06-19 EP EP18923225.9A patent/EP3663394B1/en active Active
- 2018-06-19 KR KR1020197033974A patent/KR102235141B1/ko active IP Right Grant
- 2018-06-19 JP JP2019554538A patent/JP6825182B2/ja active Active
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EP3663394B1 (en) | 2022-10-05 |
KR102235141B1 (ko) | 2021-04-05 |
US20200224169A1 (en) | 2020-07-16 |
EP3663394A1 (en) | 2020-06-10 |
WO2019241910A1 (zh) | 2019-12-26 |
KR20200002930A (ko) | 2020-01-08 |
EP3663394A4 (en) | 2021-02-24 |
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