JP2023508476A - 細胞培養基材及びその製造方法 - Google Patents
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Abstract
Description
移動刺激因子(Migration-stimulating factor、MSF)、
ミオスタチン(Myostatin、GDF-8)、神経成長因子(Nerve growth factor、NGF)、血小板由来成長因子(Platelet-derived growth factor、PDGF)、トロンボポエチン(Thrombopoietin、TPO)、T-細胞成長因子(T-cell growth factor、TCGF)、ニューロフィリン、形質転換成長因子-α(TGF-α)、形質転換成長因子-β(TGF-β)、腫瘍壊死因子-α(TNF-α)、血管内皮細胞増殖因子(VEGF)、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6及びIL-7からなる群から選ばれたいずれか1つ以上の成長因子(GF)に含まれる所定のアミノ酸配列を含んでもよい。または、ヒアルロン酸、ヘパリン硫酸塩、コンドロイチン硫酸塩、デルマタン硫酸塩、ケラタン硫酸塩、アルジン塩、フィブリン、フィブリノーゲン、コラーゲン、エラスチン、フィブロネクチン、ビトロネクチン、カドヘリン及びラミニンからなる群から選ばれたいずれか一つ以上の細胞外基質(extracellular matrix)に含まれる所定のアミノ酸の配列を含んでもよい。
基材として滅菌したポリスチレン製の6ウェルプレートを準備した。このとき、6ウェルプレートは、プラズマ処理が行われていないものを準備した。以後、下記の準備例で製造された細胞培養コーティング組成物を各ウェルあたり2mLずつピペットエイドを用いて分注した後、恒温培養器で反応して基材の表面に細胞培養コーティング層を形成させた。以後、3次蒸留水を用いて10分ずつ3回洗浄した後、クリーンベンチ内でプレート蓋を開けたまま、空気中で乾燥させて細胞培養基材を製造した。
実施例1と同様に実施して製造するが、細胞培養用融合タンパク質を配列番号14のムール貝接着タンパク質のカルボキシ末端に配列番号19の機能性ペプチドをアミノ末端に結合させたものに変更して細胞培養基材を製造した。実施例2による細胞培養コーティング層の表面SEM写真は、図2に示したように、細胞培養コーティング層が粒子が集合して形成されたことが確認できる。
実施例1と同様に実施して製造するが、細胞培養コーティング組成物でコーティングさせず、プラズマ処理も行われていない6ウェルプレートを細胞培養基材として使用した。比較例2による細胞培養コーティング層の表面SEM写真は、図3に示したように、滑らかな表面を持つことが確認できる。
実施例2と同様に実施して製造するが、細胞培養用融合タンパク質を配列番号14のムール貝接着タンパク質のカルボキシ末端に配列番号19の機能性ペプチドをアミノ末端に結合させたものに変更し、細胞培養基材の材質をポリカーボネートに変更して細胞培養基材を製造した。実施例3による細胞培養コーティング層の表面SEM写真は、図4に示したように、被コーティング基材の材質を変更した場合にも、細胞培養コーティング層が粒子が集合して形成されたことが確認できる。
実施例1と同様に実施して製造するが、細胞培養コーティング組成物において細胞培養用融合タンパク質の濃度をそれぞれ0.01mg/ml、0.1mg/ml、0.5mg/mlに変更して製造した細胞培養コーティング組成物を用いて細胞培養基材を製造した。実施例4~6による細胞培養コーティング層の表面SEM写真は、それぞれ図5、図6及び図7に示したように、細胞培養用融合タンパク質の濃度が高くなるほど形成された粒子の粒径はさらに大きくなり、粒子数が少なくなり、粒子間結合によって粒子形状が非定型の巨大粒子が形成されることが確認できる。
実施例1と同様に実施して製造するが、細胞融合タンパク質の代わりに同一濃度で配列番号15の機能性ペプチドに変更して細胞培養基材を製造した。
実施例1及び比較例2による細胞培養基材に誘導多能性幹細胞を同量分注した後、幹細胞培養培地(StemMACSTM)を用いて5日間培養後、細胞染色法で細胞培養の有無を観察し、細胞培養結果の写真を図8及び図9に示した。
実施例1による細胞培養基材においてそれぞれの培地にhiPSC-1、hESO、hiPSC-2、hiPSC-3を分注した後、mTeSR1TM、TeSR2TM、StemMACSTM、E8TM培地を用いて5日間培養した後、細胞染色法で細胞培養の有無を観察し、細胞培養結果の写真を図10に示した。
基材として滅菌されたポリスチレン製の6ウェルプレートに細胞培養用コーティング組成物として市販中のMatrigel、Vitronectin-XFTMを当該コーティング組成物メーカーのプロトコルに基づいてコーティングさせて細胞培養基材を製造した。
実施例1、比較例3及び比較例4による細胞培養基材を下記の方法により医療機器の有効期間の設定及び安定性評価によるガイドラインに従って加速老化試験を行った後、誘導多能性幹細胞を培養させて保存安定性を評価した。
Claims (10)
- 非多孔性表面を備える基材と、
前記非多孔性表面の少なくとも一部を被覆した細胞培養コーティング層と、を備え、
前記細胞培養コーティング層は、機能性ペプチドがムール貝接着タンパク質に結合された細胞培養用融合タンパク質から形成された粒子が集合したものである、細胞培養基材。 - 前記機能性ペプチドは、細胞の付着、移動、増殖及び分化のいずれか1つ以上を促進させる機能を有することを特徴とする、請求項1に記載の細胞培養基材。
- 前記基材は、ポリカーボネート、ポリスチレン、ポリイミド、ポリエステル、ポリウレタン及びガラスからなる群から選ばれたいずれか1つ以上の材質から形成されることを特徴とする、請求項1に記載の細胞培養基材。
- 前記ムール貝接着タンパク質は、配列番号1~配列番号14のアミノ酸配列からなる群から選ばれたいずれかのタンパク質または前記群から選ばれた1種以上のアミノ酸配列が連結されたタンパク質であることを特徴とする、請求項1に記載の細胞培養基材。
- 前記機能性ペプチドは、RGD配列を含むことを特徴とする、請求項1に記載の細胞培養基材。
- 前記機能性ペプチドは、配列番号15~配列番号18のアミノ酸配列からなる群から選ばれたいずれか1つ以上のペプチドまたは前記群から選ばれた1種以上のアミノ酸配列が連結されたペプチドであることを特徴とする、請求項1に記載の細胞培養基材。
- (1)カルボジイミド系カップリング剤及び反応剤を含む活性溶液と機能性ペプチドがムール貝接着タンパク質に結合された細胞培養用融合タンパク質を準備する段階と、
(2)準備された活性溶液と細胞培養用融合タンパク質を混合して細胞培養コーティング組成物を製造する段階と、
(3)細胞培養コーティング組成物を非多孔性基材の表面に処理して細胞培養コーティング層を形成させる段階と、を含む細胞培養基材の製造方法。 - 前記カルボジイミド系カップリング剤は、1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)またはN,N'-ジシクロヘキシルカルボイミド(DCC)であり、前記反応剤は、N-ヒドロキシスルホスクシンイミド(Sulfo-NHS)であることを特徴とする、請求項7に記載の細胞培養基材の製造方法。
- 前記カルボジイミドカップリング剤と反応剤は、1:0.1~10重量比で活性溶液に含まれ、
前記細胞培養コーティング組成物は、細胞培養用融合タンパク質100重量部に対してカルボジイミド系カップリング剤が1~100重量部で混合されることを特徴とする、請求項7に記載の細胞培養基材の製造方法。 - 非多孔性細胞培養基材の表面に細胞培養コーティング層を形成する細胞培養コーティング組成物であって、機能性ペプチドがムール貝接着タンパク質に結合された細胞培養用融合タンパク質、カルボジイミド系カップリング剤及び反応剤を含むことを特徴とする、非多孔性細胞培養基材用細胞培養コーティング組成物。
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