JP2015062348A - 細胞からエンドトキシンを除去する方法 - Google Patents
細胞からエンドトキシンを除去する方法 Download PDFInfo
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Abstract
Description
このような細胞培養物を臨床応用する場合には、移植を受けるレシピエントに対する安全性を確保するうえで、エンドトキシンの管理が重要となる。しかしながら、上述のような従来のエンドトキシンの除去や不活化のための手法は、医療用化合物や医療器具などの非生物を対象としたものであり、生体である細胞に適用可能な手法は未だ知られていない。
[1]細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し2回以上洗浄するステップを含む、細胞からエンドトキシンを除去する方法。
[2]洗浄するステップにおいて、細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し3〜10回洗浄する、[1]に記載の方法。
[3]洗浄するステップにおいて、細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し6回洗浄する、[2]に記載の方法。
[4]細胞凝集阻害剤が、アルブミン、トレハロース、ヒドロキシエチルデンプン、デキストラン、局所麻酔薬からなる群から選択される、[1]〜[3]のいずれかに記載の方法。
[5][1]〜[4]のいずれかに記載の方法を含む、医療用細胞培養物の製造方法。
[6][5]に記載の方法で製造された、医療用細胞培養物。
[7][6]に記載の医療用細胞培養物を含む、医薬組成物。
[8]対象において疾患を処置する方法であって、[6]に記載の細胞培養物または[7]に記載の医薬組成物の有効量をそれ必要とする対象に移植するステップを含む、前記方法。
本発明の除去方法における細胞は特に限定されず、任意の種類のものを包含するが、医療目的で使用されるものが好ましい。医療目的としては、限定されずに例えば疾患の処置、移植医療、再生医療などが挙げられる。かかる目的で使用する細胞としては、限定されずに、例えば、血液疾患などの治療に用いる造血幹細胞、免疫療法などに用いるリンパ球、樹状細胞などの免疫細胞、シート状細胞培養物を形成し得る細胞、例えば、筋芽細胞(例えば、骨格筋芽細胞など)、間葉系幹細胞(例えば、骨髄、脂肪組織、末梢血、皮膚、毛根、筋組織、子宮内膜、胎盤、臍帯血由来のものなど)、心筋細胞、線維芽細胞、心臓幹細胞、胚性幹細胞、iPS細胞、滑膜細胞、軟骨細胞、上皮細胞(例えば、口腔粘膜上皮細胞、網膜色素上皮細胞、鼻粘膜上皮細胞など)、内皮細胞(例えば、血管内皮細胞など)、肝細胞(例えば、肝実質細胞など)、膵細胞(例えば、膵島細胞など)、腎細胞、副腎細胞、歯根膜細胞、歯肉細胞、骨膜細胞、皮膚細胞等が挙げられる。
本発明において「医療用」とは、医療用途、限定されずに例えば、疾患の処置、移植医療、再生医療などの用途に使用されることを意味する。したがって、本発明の「医療用細胞培養物」は、医療製品に求められる種々の基準、例えば、エンドトキシンに関する基準などを満たすものである。かかる基準としては、例えば、行政当局によって定められたものが挙げられる。例えば、現行の第十六改正日本薬局方において、エンドトキシンの規格値はK/Mとして設定され、ここでKは発熱を誘起するといわれる体重1kg当たりのエンドトキシンの量(EU/kg)であり、投与経路による区分に基づき、脊髄腔内投与では0.2EU/kg、それ以外では5.0EU/kgと設定されており、Mは体重1kg当たり1回に投与される最大量(頻回または持続的に投与される場合、Mは1時間以内に投与される最大総量)と設定されている。したがって、60kgの成人に最大で1回に細胞培養物10mlを静脈内注射する場合、上記規格値は30EU/mlとなる。ただし、これは単なる例示であり、当業者であれば、種々の状況に適合した基準値や規格値を適宜決定することができる。なお、医療は、ヒト医療および獣医療の両方を含む。
(1)細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し2回以上洗浄するステップ、
(2)ステップ(1)で得られた細胞を凍結するステップ、
(3)凍結した細胞を解凍するステップ、
(4)シート状細胞培養物を形成するステップ、
(5)形成されたシート状細胞培養物を回収するステップ。
(1)の洗浄するステップは、本発明の除去方法についてすでに上記したとおりである。
基礎培地は、標準的な組成のまま(例えば、市販されたままの状態で)用いてもよいし、細胞種や細胞条件に応じてその組成を適宜変更してもよい。したがって、本発明に用いる基礎培地は、公知の組成のものに限定されず、1または2以上の成分が追加、除去、増量もしくは減量されたものを含む。
本発明の医薬組成物は、本発明の細胞培養物に加えて、種々の追加成分、例えば、薬学的に許容し得る担体や、細胞培養物の生存性、生着性および/または機能などを高める成分、対象疾患の処置に有用な他の有効成分などを含んでいてもよい。かかる追加成分としては、既知の任意のものを使用することができ、当業者はこれらの追加成分について精通している。また、本発明の医薬組成物は、細胞培養物の生存性、生着性および/または機能などを高める成分や、対象疾患の処置に有用な他の有効成分などと併用することができる。一態様において、本発明の医薬組成物は、組織の異常に関連する疾患の処置に用いるためのものである。処置の対象となる組織や疾患は、本発明の細胞培養物について上記したとおりである。
投与頻度は、典型的には1回の処置につき1回であるが、所望の効果が得られない場合には、複数回投与することも可能である。
2.5gのヒトアルブミン(一般社団法人日本血液製剤機構製)を500mLのハンクス平衡塩液に加え、ヒトアルブミンを0.5w/v%の濃度で含む洗浄液を調製した。3×107個のヒト骨格筋芽細胞を30mLの洗浄液に懸濁した細胞懸濁液を50mLコニカルチューブに入れ、そこにエンドトキシン(E. coli UKT-B株由来、和光純薬工業(株)製)を添加した。細胞懸濁液を100μL採取し、その後エンドトキシン量の測定に供した。細胞懸濁液を240×gで7分間遠心して上清を廃棄した。チューブに洗浄液を28.5mL加え、ピペッティングにより細胞を十分に撹拌した後、240×gで7分間遠心して上清を廃棄した。この操作を6回繰り返した。6回目の洗浄操作で、上清を廃棄した後に残った細胞を3mLの洗浄液に懸濁し、エンドトキシン量の測定に供した。また、未使用の洗浄液もエンドトキシン量の測定に供した。なお、洗浄液には、一般的な作業条件下で混入する可能性のあるエンドトキシンの影響を評価するために、予め一定量のエンドトキシンを添加しておいた。各検体のエンドトキシン量は、トキシノメーターET−6000およびリムルスES−IIシングルテストワコー(いずれも和光純薬工業(株)製)を用いて比濁時間法(カイネティック−比濁法)にて測定した。結果を下表に示す。
Claims (8)
- 細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し2回以上洗浄するステップを含む、細胞からエンドトキシンを除去する方法。
- 洗浄するステップにおいて、細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し3〜10回洗浄する、請求項1に記載の方法。
- 洗浄するステップにおいて、細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し6回洗浄する、請求項2に記載の方法。
- 細胞凝集阻害剤が、アルブミン、トレハロース、ヒドロキシエチルデンプン、デキストラン、局所麻酔薬からなる群から選択される、請求項1〜3のいずれか一項に記載の方法。
- 請求項1〜4のいずれか一項に記載の方法を含む、医療用細胞培養物の製造方法。
- 請求項5に記載の方法で製造された、医療用細胞培養物。
- 請求項6に記載の医療用細胞培養物を含む、医薬組成物。
- 対象において疾患を処置する方法であって、請求項6に記載の細胞培養物または請求項7に記載の医薬組成物の有効量をそれ必要とする対象に投与するステップを含む、前記方法。
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CN111606978A (zh) * | 2020-06-09 | 2020-09-01 | 河南省农业科学院 | 一种去除大肠杆菌重组表达猪细小病毒vp2蛋白中内毒素的方法 |
CN113092767A (zh) * | 2021-04-02 | 2021-07-09 | 丹娜(天津)生物科技股份有限公司 | 一种鲎试剂冻干微球及其制备方法和应用 |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111606978A (zh) * | 2020-06-09 | 2020-09-01 | 河南省农业科学院 | 一种去除大肠杆菌重组表达猪细小病毒vp2蛋白中内毒素的方法 |
CN111606978B (zh) * | 2020-06-09 | 2023-03-17 | 河南省农业科学院 | 一种去除大肠杆菌重组表达猪细小病毒vp2蛋白中内毒素的方法 |
CN113092767A (zh) * | 2021-04-02 | 2021-07-09 | 丹娜(天津)生物科技股份有限公司 | 一种鲎试剂冻干微球及其制备方法和应用 |
CN113092767B (zh) * | 2021-04-02 | 2024-03-12 | 丹娜(天津)生物科技股份有限公司 | 一种鲎试剂冻干微球及其制备方法和应用 |
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