JP2016202164A - 造血幹細胞/前駆細胞の製造方法、及び、造血幹細胞/前駆細胞の組成物 - Google Patents
造血幹細胞/前駆細胞の製造方法、及び、造血幹細胞/前駆細胞の組成物 Download PDFInfo
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Abstract
Description
本発明の製造方法によって製造された高純度の造血幹細胞/前駆細胞は、高い比率で臨床的に有効な造血幹細胞/前駆細胞(CD34+CD38-細胞)を含むことのみならず、その精製及び製造過程において動物由来の成分を含有する試薬を使用しないため、得られた造血幹細胞/前駆細胞の培養物は臨床へ直接に応用することができる。
を一夜培養した組では、造血幹細胞/前駆細胞の純度が76.2%である。また、本発明の好ましい実施例では、造血幹細胞/前駆細胞の純度が92%である。上述の実験が説明した通り、本発明の単球を一夜培養する方法は、解凍により損傷された単球の活性を回復することができる。よって、造血幹細胞/前駆細胞の収穫純度を高めることができる。
(1)第一組:5ng/mLのIL−3、10ng/mLのIL−6、50ng/mLのSCF、20ng/mLのFLT−3L、15nMのTAT−HOXB4を含む;
(2)第二組:5ng/mLのIL−3、10ng/mLのIL−6、100ng/mLのSCF、20ng/mLのFLT−3L、15nMのTAT−HOXB4を含む;
(3)第三組:5ng/mLのIL−3、10ng/mLのIL−6、100ng/mLのSCF、20ng/mLのFLT−3L、25ng/mLのTPO、15nMのTAT−HOXB4を含む。
(1)第一組:1x104cells/mLの細胞密度で7日培養する。
(2)第二組:5x104cells/mLの細胞密度で3日培養し、第4日目から、細胞密度を1.5x104cells/mLに変更し、第7日まで培養する。
(3)第三組:5x104cells/mLの細胞密度で3日培養し、第4日目から、細胞密度を3x104cells/mLに変更し、第7日まで培養する。
(4)第四組:5x104cells/mLの細胞密度で7日培養する。
(1)調合法1:80%のAlbuminar・−25,20%のCryoSure−DEX40(20%の人アルブミンを含む);
(2)調合法2:48%のAlbuminar・−25,20%のCryoSure−DEX40,生理食塩水(12%の人アルブミンを含む);
(3)調合法3:24%のAlbuminar・−25,20%のCryoSure−DEX40,生理食塩水(6%の人アルブミンを含む)。
Claims (11)
- 生体外で培養を行い、高度に精製されており、かつ直ちに使用可能な造血幹細胞/前駆細胞の製造方法であって、
造血幹細胞/前駆細胞を含む血液を解凍し、密度勾配遠心分離により単球の精製を行い、高純度の単球を得るステップと、
得られた高純度の単球を一夜培養し、造血幹細胞/前駆細胞を分離するステップと、
分離された造血幹細胞/前駆細胞を、サイトカインおよびTAT−HOXB4を含むIMDM/5%HABS培養液で4から7日培養するステップと、
造血幹細胞/前駆細胞の培養物を収穫するステップと、を含むことを特徴とする造血幹細胞/前駆細胞の製造方法。 - 24−80%のAlbuminar(登録商標)・−25、および、6−20%の人アルブミンを含む20%のCryoSure(登録商標)−DEX40を含む保存材で、収穫した造血幹細胞/前駆細胞を冷凍保存するステップをさらに含むことを特徴とする請求項1に記載の造血幹細胞/前駆細胞の製造方法。
- 前記一夜培養は、IMDM/5%HABS培養液を用いて、5x105−6x106cells/mLの細胞密度範囲内で、単球を16〜18時間培養することであることを特徴とする請求項1に記載の造血幹細胞/前駆細胞の製造方法。
- 前記サイトカインは、インターロイキン−3、インターロイキン−6、幹細胞因子、FLT−3ゲニン、および、トロンボポエチンを含むことを特徴とする請求項1に記載の造血幹細胞/前駆細胞の製造方法。
- 前記サイトカインは、5〜10ng/mLのIL−3、10〜20ng/mLのIL−6、50〜100ng/mLのSCF、20〜40ng/mLのFLT−3L、および25〜50ng/mLのTPOを含むことを特徴とする請求項1に記載の造血幹細胞/前駆細胞の製造方法。
- 分離された造血幹細胞/前駆細胞を、サイトカインおよびTAT−HOXB4を含むIMDM/5%HABS培養液を用いて、1x104−5x105cells/mLの細胞密度範囲内で培養することを特徴とする請求項1に記載の造血幹細胞/前駆細胞の製造方法。
- 前記保存材は、80%のAlbuminar・−25、および、20%の人アルブミンを含む20%のCryoSure−DEX40からなることを特徴とする請求項2に記載の造血幹細胞/前駆細胞の製造方法。
- 請求項1に記載の製造方法により製造された造血幹細胞/前駆細胞の組成物であって、
臨床で有効な造血幹細胞/前駆細胞(CD34+CD38-細胞)を15〜40%含むことを特徴とする組成物。 - 臨床で有効な造血幹細胞/前駆細胞(CD34+CD38-細胞)を25〜30%含むことを特徴とする請求項8に記載の組成物。
- 造血幹細胞/前駆細胞を含む前記血液は、臍帯血であることを特徴とする請求項1に記載の造血幹細胞/前駆細胞の製造方法。
- 造血幹細胞/前駆細胞を含む前記血液は、末梢血であることを特徴とする請求項1に記載の造血幹細胞/前駆細胞の製造方法。
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- 2015-06-08 JP JP2015115770A patent/JP6348881B2/ja active Active
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US20160304837A1 (en) | 2016-10-20 |
CN106190979A (zh) | 2016-12-07 |
CN106190979B (zh) | 2020-04-03 |
JP6348881B2 (ja) | 2018-06-27 |
TW201638332A (zh) | 2016-11-01 |
TWI548748B (zh) | 2016-09-11 |
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