JP5698674B2 - 治療用細胞組成物 - Google Patents
治療用細胞組成物 Download PDFInfo
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- JP5698674B2 JP5698674B2 JP2011539102A JP2011539102A JP5698674B2 JP 5698674 B2 JP5698674 B2 JP 5698674B2 JP 2011539102 A JP2011539102 A JP 2011539102A JP 2011539102 A JP2011539102 A JP 2011539102A JP 5698674 B2 JP5698674 B2 JP 5698674B2
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Description
(i)トロロックス、Na+、K+、Ca2+、Mg2+、Cl−、H2PO4 −、HEPES、ラクトビオネート、ショ糖、マンニトール、グルコース、デキストラン−40、アデノシン、およびグルタチオン;ならびに
(ii)幹細胞または前駆細胞
を含み、かつ、前記組成物は、双極性非プロトン性溶媒(特にDMSO)を含まない。
(a)本発明の第1の態様の組成物中で前記細胞を懸濁する工程;
(b)前記工程(a)の前記細胞懸濁物を凍結温度(cryothermic
temperature)で保存する工程;および
(c)前記工程(b)の前記細胞懸濁物を解凍する工程。
(a)HTS−FRS中で前記細胞を懸濁する工程;
(b)前記工程(a)の懸濁させた前記細胞を凍結温度で保存する工程;および
(c)前記工程(b)の懸濁させた前記細胞を解凍する工程。
(神経幹細胞、網膜前駆細胞および間葉幹細胞の製剤化)
神経幹細胞はヒト胎児脳由来であり、Polock et al(2006)によって記載されているように、組織培地内に保持された。網膜前駆細胞培地は胎児網膜から得られ、Aftab et al(2009)によって記載されているように、組織培地中に保持された。健常志願者の骨盤骨の後腸骨稜から抜き取られた骨髄から単離されたヒト間葉幹細胞をLonza(カタログ番号:PT−2501)から入手し、製造者所有の培地であるMSCGM(間葉成長培地)を用いて製造者が推薦するように培養した。2代培養で供給された細胞を、トリプシン/EDTAを用いて3代培養し、上述した製剤化の前の初期捲種密度を5000〜6000細胞/cm2とした。
HypoThermosol(登録商標)-FRS中で製剤化された神経幹細胞の代謝活性は、生理食塩水溶液中で製剤化された神経肝細胞の代謝活性と同程度である。HypoThermosol(登録商標)-FRSまたは生理食塩水中の細胞製剤を1時間保存し、次いで代謝活性アッセイ(Dojindo CCK-8)を培地中で1時間行った。存在する細胞の数を考慮して、細胞定量化アッセイ(Invitrogen CyQUANT)を用いて結果を標準化した。データは、HypoThermosol(登録商標)-FRS中で製剤化された細胞が、生理食塩水中で製剤化された細胞の代謝活性と同程度の代謝活性を有することを示している(図1参照)。
品質保持期限の試験は、生理食塩水中で製剤化されるかまたはHTS−FRSを用いる本発明で記載された方法を用いて、治療用および市販用に製造された神経幹細胞株であるCTX0E03(Pollock et al., 2006)の生存能力を評価している。一般に、前記細胞は投与前に2℃〜8℃で保存されるだろう。しかしながら、前記細胞の保存条件は、投与の間は周囲温度であるだろうし、かつ、品質保持期限アッセイの間、この温度変化が考慮される。2℃〜8℃で5時間保存し次いで周囲温度への温度変化を行ったすべての実験からのデータが図2に示されており、該データは、HTS−FRSによって生じた、生理食塩水製剤を超える、生存可能な品質保持期限の増加を明らかに実証する。同じ培地由来の細胞との処理の比較では、HBSS+NAC処理後のHTS−FRS処理によって生じた、7時間の時点の生存能力における平均増加は22.7%である(HBSS+NACの生存能力=58.9%である;HTS−FRSの生存能力=81.6%である)。今までの各場合において、本発明のHTS−FRS製剤の生存能力は、生存可能な細胞産物に対する調節信頼性によって定められているように、上述の70%を超える許容基準になっている。
神経幹細胞、網膜前駆細胞および間葉幹細胞の凍結保存は、本発明の方法を用いて首尾よく達成されている。本発明の方法にしたがって製剤化された細胞は、細胞生存能力が悪化することなく、−80℃での保存が最大4日間可能である(図7ないし図10参照)。HTS−FRS非毒性溶液は、10%DMSOを含む現行の方法を用いて達成されるのと同様に、続いて凍結保存が行われる細胞の生存能力を維持する点で効果的であった(図8ないし図10)。加えて、解凍した本発明の細胞は、続くHTS−FRS中での解凍においても生存能力を維持している(図8ないし図10参照)。さらに、これらの解凍した細胞は、組織培地中に配置したときに、10%DMSOを用いて凍結保存された細胞と同等の生物学的活性を示している。
細胞治療製剤の投与に関して許容されうる賦形剤の鍵となる起因のひとつは、クリニックで使用されるであろう外科装置との互換性である。本明細書で記載された方法にしたがって製剤化された細胞療法の粘度および密度の違いならびに生理食塩水溶液は、理論的には、シリンジおよび細胞伝達カニューレを介して細胞を運ぶビヒクルの能力に影響を及ぼすであろう。しかしながら、表2に示されるように、HypoThermosol(登録商標)-FRS中で製剤化された細胞は、生理食塩水中で製剤化された細胞と同じ成功率で、細胞伝達カニューレを通過する。細胞製剤(50,000細胞/μl)が250μlのガラス製シリンジに引き込まれ、200μl が1μl/分または5μl/分の速度で19cm細胞伝達カニューレを介して放出された。細胞の生存能力をトリパンブルー排除(exclusion)によって評価し、かつ、細胞の濃度を血球計を用いて測定した。加えて、前記細胞のネスチン免疫反応性を、蛍光抗体およびフローサイトメトリーを用いて測定した。生存能力は、各製剤において許容範囲にあった。細胞濃度は一定である。ネスチン免疫反応性は、上述の予め定められた93%の下限を超えたままであった。これらの結果はさらに、賦形剤としてのHTS−FRSの使用を実証している。
細胞試料を0.4%トリパンブルー(Sigma)と1:1で混合し、血球計の上に配置した。生存能力がある細胞は細胞質から染料を排除し、無色である。細胞膜の完全性を失っている生存能力がない細胞は、青に染色される。試料の生存能力および濃度は、10倍の対象位相差顕微鏡分析を用いて、血球計のマス目内で細胞を計測することにより決定される。
本発明における方法によって製剤化された細胞療法の毒性は変化しないだろう。マウスへと移植された場合、HypoThermosol(登録商標)-FRSビヒクルまたは神経幹細胞とともに想定される明らかな毒性は認められなかった。これらの試験は頭蓋内投与および筋肉内投与を含む。加えて、毒性試験はラットについても完了しており、HypoThermosol(登録商標)-FRS溶液または生理食塩水溶液の頭蓋内投与に対する被験者の応答の違いや、いずれかの溶液に対する明らかな反応は示されなかった。
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Claims (18)
- (i)トロロックス、Na+、K+、Ca2+、Mg2+、Cl−、H2PO4 −、HEPES、ラクトビオネート、ショ糖、マンニトール、グルコース、デキストラン−40、アデノシン、およびグルタチオン;ならびに
(ii)神経幹細胞、間葉幹細胞、網膜幹細胞、またはこれらの前駆細胞
を含み、かつ、双極性非プロトン性溶媒を含まない、治療用組成物であって、
患者への直接投与に使用され、かつ、以下の工程(a)ないし(c)によって製造される、治療用組成物。
(a)トロロックス、Na + 、K + 、Ca 2+ 、Mg 2+ 、Cl − 、H 2 PO 4 − 、HEPES、ラクトビオネート、ショ糖、マンニトール、グルコース、デキストラン−40、アデノシン、およびグルタチオンを含み、かつ、双極性非プロトン性溶媒を含まない組成物中で、神経幹細胞、間葉幹細胞、網膜幹細胞、またはこれらの前駆細胞を懸濁する工程;
(b)前記工程(a)の前記細胞懸濁物を凍結温度で保存する工程;および
(c)前記工程(b)の前記細胞懸濁物を解凍する工程。 - 前記神経幹細胞、前記間葉幹細胞、および前記網膜幹細胞は、ヒト幹細胞であり、
前記前駆細胞は、ヒト前駆細胞である、請求項1に記載の組成物。 - 前記細胞は、生分解性高分子物質に含まれる、請求項1または2に記載の組成物。
- 前記生分解性高分子物質はPLGAである、請求項3に記載の組成物。
- (i)トロロックス、Na+、K+、Ca2+、Mg2+、Cl−、H2PO4 −、HEPES、ラクトビオネート、ショ糖、マンニトール、グルコース、デキストラン−40、アデノシン、およびグルタチオンを含み、かつ、双極性非プロトン性溶媒を含まない組成物中で、神経幹細胞、間葉幹細胞、網膜幹細胞、またはこれらの前駆細胞を懸濁することを含む、患者への投与のために神経幹細胞、間葉幹細胞、網膜幹細胞、またはこれらの前駆細胞を製剤化する方法であって、
下記の工程(a)ないし(c)を含む、方法:
(a)前記(i)組成物中で、前記神経幹細胞、前記間葉幹細胞、前記網膜幹細胞、または前記前駆細胞を懸濁する工程;
(b)前記工程(a)の前記細胞懸濁物を凍結温度で保存する工程;および
(c)前記工程(b)の前記細胞懸濁物を解凍する工程。 - 前記神経幹細胞、前記間葉幹細胞、前記網膜幹細胞、または前記前駆細胞は、細胞培養系から回収される、請求項5に記載の方法。
- 前記工程(b)は、−70℃〜−200℃の温度で行われる、請求項5に記載の方法。
- 前記工程(b)は、−80℃〜−196℃の温度で行われる、請求項7に記載の方法。
- 前記細胞懸濁物は、前記工程(b)または前記工程(c)の後で低温度で保存される、請求項5ないし8のいずれか1項に記載の方法。
- 前記低温度での保存は2℃〜8℃で行われる、請求項9に記載の方法。
- 前記神経幹細胞、前記間葉幹細胞、および前記網膜幹細胞は、ヒト幹細胞であり、
前記前駆細胞は、ヒト前駆細胞である、請求項5ないし10のいずれか1項に記載の方法。 - 前記神経幹細胞、前記間葉幹細胞、前記網膜幹細胞、または前記前駆細胞は、生分解性高分子物質に含まれる、請求項5ないし11のいずれか1項に記載の方法。
- 前記生分解性高分子物質はPLGAである、請求項12に記載の方法。
- 前記神経幹細胞、前記間葉幹細胞、前記網膜幹細胞、または前記前駆細胞はカプセルに包含されている、請求項5ないし13のいずれか1項に記載の方法。
- 前記神経幹細胞、前記間葉幹細胞、前記網膜幹細胞、または前記前駆細胞は、アルギン酸塩、キトサン、PEG、PLL、ポリ−L−オルニチン14、ポリ(メチレン−co−グアジニン)塩酸塩、グリセリンリン酸、ヒアルロン酸、セルロースリン酸、澱粉、寒天、カラギーナン、絹フィブロイン、ゼラチンおよびジェランガムから選ばれる物質に包含されている、請求項14に記載の方法。
- 前記製剤化された前記神経幹細胞、前記間葉幹細胞、前記網膜幹細胞、または前記前駆細胞は、細胞伝達カニューレを介した投与が可能である、請求項7ないし15のいずれか1項に記載の方法。
- 請求項1ないし4のいずれか1項に記載の組成物を単位用量の形態で含む、薬剤。
- 前記薬剤は、組織への移植または全身伝達を介して、該薬剤を必要とする患者に直接投与される、請求項17に記載の薬剤。
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