JP7065542B1 - 細胞の生産方法 - Google Patents
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Abstract
Description
iPS細胞(253G1株)について、iMatrix-511(ニッピ)でコーティングした10cmディッシュを用いて、Essential 8培地(サーモフィッシャー)中で接着培養して増殖継代を行った後、0.5mM EDTA溶液で10分間処理し、剥離したiPS細胞を実験に用いた。剥離したiPS細胞を7x10e4/mlの細胞濃度になるようにEssential 8培地に懸濁し、直径6cm(面積20cm2)円型ディッシュ(ファルコン)、直径10cm(面積50cm2)円型ディッシュ(ファルコン)、直径15cm(面積150cm2)円型ディッシュ(ファルコン)、1辺22cm(面積500cm2)正方形型ディッシュ(住友ベークライト、品番MS-12450)に、0.4ml/cm2の液量になるように、それぞれ8ml、20ml、60ml、200mlの細胞懸濁液を添加して、37℃、5%CO2のインキュベーター内で静置培養あるいは旋回培養を行った。旋回培養については、インキュベーター内用シェイカー(オプティマ、OS-762RC)を用いて、30、50、60rpmで旋回培養(旋回径25mm)を行った。計4日間培養した後、浮遊しているiPS細胞塊をトリプシン溶液で分散させてセルカウントを行い、総細胞数(各ディッシュあたり)と細胞増殖率(4日後の細胞数/播種時の細胞数)を計算した。
各ディッシュについて、静置培養を行った場合、全てのiPS細胞がディッシュに接着し、浮遊しているiPS細胞は得られなかった(図1、2)。30、50、60rpmで旋回培養を行ったところ、各ディッシュで浮遊しているiPS細胞塊が得られた。全ての条件で円型のディッシュに比べ正方形型ディッシュの方が細胞増殖率が高く(最大40倍)、トータルの細胞数も多い(最大3x10e8/ディッシュ)傾向が見られた(図1、2)。さらに、円型ディッシュと正方形型ディッシュで50rpm旋回培養したiPS細胞の心筋分化能を評価したところ、正方形型ディッシュの方が心筋細胞マーカーであるトロポニンTの陽性細胞の割合が高い、つまり心筋細胞の純度が高いことがわかった(図3)。
Claims (8)
- 細胞及び培地を収容する容器を水平回転させる工程を含み、前記容器の底面形状は四角型の形状であり、前記細胞はiPS細胞又は間葉系幹細胞である、細胞の生産方法。
- 前記細胞は、浮遊細胞である、請求項1に記載の生産方法。
- 前記細胞は、細胞塊、又はプラスチック素材と接着した細胞である、請求項1又は2に記載の生産方法。
- 前記細胞を浮遊培養する工程を含む、請求項1~3のいずれかに記載の生産方法。
- 細胞及び酵素液を収容する容器を水平回転させる工程を含み、前記容器の底面形状は四角型の形状であり、前記細胞はiPS細胞又は間葉系幹細胞である、細胞の酵素処理方法。
- 前記細胞は、細胞塊又はプラスチック素材と接着した細胞である、請求項5に記載の酵素処理方法。
- 前記酵素液は、細胞塊又は接着細胞を単一細胞に分散処理するための蛋白質分解酵素を含む溶液である、請求項5又は6に記載の酵素処理方法。
- 前記細胞を浮遊培養する工程を含む、請求項5~7のいずれかに記載の酵素処理方法。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2008212049A (ja) | 2007-03-02 | 2008-09-18 | Fujimori Kogyo Co Ltd | 培養袋及び培養装置 |
JP2015509701A (ja) | 2011-12-02 | 2015-04-02 | アーメイゲン・テクノロジーズ・インコーポレイテッドArmagen Technologies, Inc. | Cnsにおけるアリールスルファターゼa活性を増加するための方法および組成物 |
WO2017094451A1 (ja) | 2015-12-04 | 2017-06-08 | 公立大学法人大阪府立大学 | 細胞培養容器及び観察用試料セル |
US20180147552A1 (en) | 2016-11-28 | 2018-05-31 | Sarfaraz K. Niazi | Zero gravity process device |
JP2019198850A (ja) | 2018-05-18 | 2019-11-21 | 藤森工業株式会社 | 撹拌容器および撹拌方法 |
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JP2021027821A (ja) * | 2019-08-09 | 2021-02-25 | 宇部興産株式会社 | 細胞培養装置及びそれを使用した細胞培養方法 |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2008212049A (ja) | 2007-03-02 | 2008-09-18 | Fujimori Kogyo Co Ltd | 培養袋及び培養装置 |
JP2015509701A (ja) | 2011-12-02 | 2015-04-02 | アーメイゲン・テクノロジーズ・インコーポレイテッドArmagen Technologies, Inc. | Cnsにおけるアリールスルファターゼa活性を増加するための方法および組成物 |
WO2017094451A1 (ja) | 2015-12-04 | 2017-06-08 | 公立大学法人大阪府立大学 | 細胞培養容器及び観察用試料セル |
US20180147552A1 (en) | 2016-11-28 | 2018-05-31 | Sarfaraz K. Niazi | Zero gravity process device |
JP2019198850A (ja) | 2018-05-18 | 2019-11-21 | 藤森工業株式会社 | 撹拌容器および撹拌方法 |
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