JPWO2019131942A1 - 細胞凝集促進剤 - Google Patents
細胞凝集促進剤 Download PDFInfo
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- JPWO2019131942A1 JPWO2019131942A1 JP2019562204A JP2019562204A JPWO2019131942A1 JP WO2019131942 A1 JPWO2019131942 A1 JP WO2019131942A1 JP 2019562204 A JP2019562204 A JP 2019562204A JP 2019562204 A JP2019562204 A JP 2019562204A JP WO2019131942 A1 JPWO2019131942 A1 JP WO2019131942A1
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Abstract
Description
(1)SRF阻害剤を含む、細胞の浮遊培養に用いるための細胞凝集促進剤。
(2)前記SRF阻害剤の濃度が9.0μg/mL以上10.0mg/mL以下である、(1)に記載の細胞凝集促進剤。
(3)さらにROCK阻害剤を含む、(1)又は(2)に記載の細胞凝集促進剤。
(4)前記SRF阻害剤がCCG−1423である、(1)から(3)のいずれかに記載の細胞凝集促進剤。
(5)前記細胞が幹細胞である、(1)から(4)のいずれかに記載の細胞凝集促進剤。
(6)SRF阻害剤を含む培地中で細胞を浮遊培養する工程を含む、細胞凝集塊の製造方法。
(7)前記培地中における前記SRF阻害剤の濃度が4.5ng/mL以上4.6mg/mL以下である、(6)に記載の製造方法。
(8)さらに、前記培地中にROCK阻害剤を含む、(6)又は(7)に記載の製造方法。
(9)前記SRF阻害剤がCCG−1423である、(6)から(8)のいずれかに記載の製造方法。
(10)前記細胞が幹細胞である、(6)から(9)のいずれかに記載の製造方法。
(11)(6)から(10)のいずれかに記載の製造方法により得られた細胞凝集塊。
(12)細胞と、培地と、SRF阻害剤とを含む、細胞培養組成物。
(13)前記SRF阻害剤の濃度が4.5ng/mL以上4.6mg/mL以下である、(12)に記載の細胞培養組成物。
(14)さらにROCK阻害剤を含む、(12)又は(13)に記載の細胞培養組成物。
(15)前記SRF阻害剤がCCG−1423である、(12)から(14)のいずれかに記載の細胞培養組成物。
(16)前記細胞が幹細胞である、(12)から(15)のいずれかに記載の細胞培養組成物。
(17)前記細胞の形態が細胞凝集塊である、(12)から(16)のいずれかに記載の細胞培養組成物。
(18)SRF阻害剤を含む培地中で細胞を浮遊培養する工程を含む、細胞の凝集を促進する方法。
(19)前記培地中における前記SRF阻害剤の濃度が4.5ng/mL以上4.6mg/mL以下である、(18)に記載の方法。
(20)さらにROCK阻害剤を含む、(18)又は(19)に記載の方法。
(21)前記SRF阻害剤がCCG−1423である、(18)から(20)のいずれかに記載の方法。
(22)前記細胞が幹細胞である、(18)から(21)のいずれかに記載の方法。
(23)培地と、SRF阻害剤とを含む、細胞培養培地。
(24)前記SRF阻害剤の濃度が4.5ng/mL以上4.6mg/mL以下である、(23)に記載の細胞培養培地。
(25)さらにROCK阻害剤を含む、(23)又は(24)に記載の細胞培養培地。
(26)さらに増殖因子を含む、(23)から(25)のいずれかに記載の細胞培養培地。
(27)幹細胞の培養に用いるための、(23)から(26)のいずれかに記載の細胞培養培地。
(28)細胞凝集塊を作製するための、(23)から(27)のいずれかに記載の細胞培養培地。
(29)細胞凝集塊の70%以上(重量基準)において、最も幅の広い部分の寸法が500μm以下、好ましくは300μm以下である、(6)から(10)のいずれかに記載の製造方法、(11)に記載の細胞凝集塊、(17)に記載の細胞培養組成物、又は(28)に記載の細胞培養培地。
(30)細胞凝集塊の70%以上(重量基準)において、最も幅の広い部分の寸法が40μm以上、好ましくは100μm以上である、(6)から(10)及び(29)のいずれかに記載の製造方法、(11)又は(29)に記載の細胞凝集塊、(17)又は(29)に記載の細胞培養組成物、又は(28)もしくは(29)に記載の細胞培養培地。
本発明において細胞凝集塊を形成する細胞は、接着性を有する細胞(接着性細胞)であることができる。接着性細胞は、動物由来細胞等であることができ、好ましくは哺乳類動物由来細胞等であることができ、より好ましくは生体組織由来細胞及び生体組織由来細胞から派生した細胞等であることができ、特に好ましくは上皮組織由来細胞及び上皮組織細胞から派生した細胞等、又は結合組織由来細胞及び結合組織由来細胞から派生した細胞等、又は筋組織由来細胞及び筋組織由来細胞から派生した細胞等、又は神経組織由来細胞及び神経組織由来細胞から派生した細胞等であることができ、さらに好ましくは動物由来幹細胞及び動物由来幹細胞から分化した細胞等であることができ、もっと好ましくは動物由来多能性幹細胞及び動物由来多能性幹細胞から分化した細胞等であることができ、よりさらに好ましくは哺乳類動物由来多能性幹細胞及び哺乳類動物由来多能性幹細胞から分化した細胞等であることができ、もっとも好ましくはヒト由来多能性幹細胞及びヒト由来多能性幹細胞から分化した細胞等であることができる。
細胞凝集塊は、複数の細胞が三次元的に凝集して形成される塊状の細胞集団であって、スフェロイドとも呼ばれる。細胞凝集塊は、典型的には、概ね球状の形状を有する。
本発明で用いる培地は、任意の動物細胞培養用培地を基礎培地とし、SRF阻害剤若しくはSRF阻害剤とROCK阻害剤、又はSRF阻害剤若しくはSRF阻害剤とROCK阻害剤を含む細胞凝集促進剤、及び、必要に応じて他の成分を適宜添加することにより調製することができる。本発明で用いる培地は細胞の浮遊培養に適したものであることが好ましく、典型的には、液体培地である。
SRF阻害剤は、MADS(MCM1, Agamous, Deficiens, and SRF)ボックススーパーファミリーに属する転写因子である血清応答因子(SRF、serum response factor)の活性を阻害する物質として定義され、例えば、CCG−1423(N−[2−[4(4−クロロフェニル)アミノ]−1−メチル−2−オキソエトキシ]−3,5−ビス(トリフルオロメチル)−ベンズアミド)、CCG−1423アナログであるCCG−100602(1−[3,5−ビス(トリフルオロメチル)ベンゾイル]−N−(4−クロロフェニル)−3−ピペリジンカルボキサミド)、CCG−203971(6−amino−1,4−dihydro−1,3−dimethyl−4−[4−(trifluoromethyl)phenyl]−pyrano[2,3−c]pyrazole−5−carbonitrile)、CCG−222740(参考文献:Yu−Wai−Man C et al. Local delivery of novel MRTF/SRF inhibitors prevents scar tissue formation in a preclinical model of fibrosis. Sci Rep. 2017 Mar 31;7(1):518)及びそれらの誘導体、並びにSRFに対するアンチセンス核酸、RNA干渉誘導性核酸(例えば、siRNA)、ドミナントネガティブ変異体、及びそれらの発現ベクターが挙げられる。
ROCK阻害剤は、Rho−キナーゼ(ROCK,Rho−associated protein kinase)のキナーゼ活性を阻害する物質として定義され、例えば、Y−27632(4−[(1R)−1−アミノエチル]−N−ピリジン−4−イルシクロヘキサン−1−カルボキサミド又はその塩(例えば2塩酸塩))(例えば、Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000);Narumiya et al., Methods Enzymol. 325,273-284 (2000)参照)、H−1152((S)−(+)−2−メチル−1−[(4−メチル−5−イソキノリニル)スルホニル]−ヘキサヒドロ−1H−1,4−ジアゼピン又はその塩(例えば2塩酸塩))(例えば、Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)参照)、Fasudil/HA1077(1−(5−イソキノリンスルホニル)ホモピペラジン又はその塩(例えば2塩酸塩))(例えば、Uenata et al., Nature 389: 990-994 (1997)参照)、Wf−536((+)−(R)−4−(1−アミノエチル)−N−(4−ピリジル)ベンズアミド1塩酸塩)(例えば、Nakajima et al., CancerChemother. Pharmacol. 52(4): 319-324 (2003)参照)、Y39983(4−[(1R)−1−Aminoethyl]−N−1H−pyrrolo[2、3−b]pyridin−4-ylbenzamide dihydrochloride)、SLx−2119(2−[3−[4−(1H−indazol−5−ylamino)−2−quinazolinyl]phenoxy]−N−(1-methylethyl)−acetamide)、Azabenzimidazole−aminofurazans、DE−104、XD−4000、HMN−1152、4−(1−aminoalkyl)−N−(4−pyridyl)cyclohexane−carboxamides、Rhostatin、BA−210、BA−207, BA−215, BA−285, BA−1037、Ki−23095、VAS−012(例えば、James K. Liao et al., J Cardiovasc Pharmacol. 2007 Jul;50(1):17-24.)及びそれらの誘導体、並びにROCKに対するアンチセンス核酸、RNA干渉誘導性核酸(例えば、siRNA)、ドミナントネガティブ変異体、及びそれらの発現ベクターが挙げられる。また、ROCK阻害剤としては他の低分子化合物も知られており、本発明においてはこのような化合物又はそれらの誘導体もROCK阻害剤として使用できる(例えば、米国特許出願公開第20050209261号、同第20050192304号、同第20040014755号、同第20040002508号、同第20040002507号、同第20030125344号、同第20030087919号、及び国際公開第2003/062227号、同第2003/059913号、同第2003/062225号、同第2002/076976号、同第2004/039796号参照)。ROCK阻害剤としては、1種又は2種以上のROCK阻害剤を使用することができる。
本発明の一態様は、SRF阻害剤、又はSRF阻害剤及びROCK阻害剤を含む培地中で細胞を浮遊培養する工程(浮遊培養工程)を含む、細胞の凝集を促進する方法である。
SRF阻害剤、又はSRF阻害剤及びROCK阻害剤を含む培地中で細胞を浮遊培養する工程(浮遊培養工程)の具体的な実施形態について説明する。
「維持培養工程」は、浮遊培養工程前の細胞集団、又は浮遊培養工程後、若しくはその後の回収工程後に得られる細胞凝集塊を、未分化性を維持した状態で細胞を増殖させるために培養する工程である。維持培養は、細胞を容器、担体等培養基材に接着させながら培養する接着培養であってもよいし、細胞を培地中で浮遊させながら培養する浮遊培養であってもよい。
「回収工程」は、維持培養工程又は浮遊培養工程後の培養液から培養した細胞を回収する工程で、本発明の方法における選択工程である。
細胞を浮遊培養法で培養した場合、細胞は培養液中に浮遊した状態で存在する。したがって、細胞の回収は、静置状態又は遠心分離により上清の液体成分を除去することで達成できる。また、細胞の回収方法としてはフィルターや中空糸分離膜等を選択することもできる。
接着培養法で細胞を培養した場合、培養後、多くの細胞は培養容器や培養担体等の外部マトリクスに接着した状態で存在する。したがって、培養容器から培養液を除去するには、培養後の容器を静かに傾けて液体成分を流し出せばよい。外部マトリクスに接着した細胞が培養容器内に残るため、培養液と細胞を容易に分離することができる。
本明細書において「単一細胞化」とは、単層細胞片や細胞凝集塊等のように複数の細胞が互いに接着又は凝集した細胞集合体を分散させて、単一の遊離した細胞状態にすることをいう。
本発明の他の一態様は、SRF阻害剤、又はSRF阻害剤及びROCK阻害剤を含む、細胞の浮遊培養に用いるための細胞凝集促進剤である。
本発明の他の一態様は、SRF阻害剤、又はSRF阻害剤及びROCK阻害剤を含む培地中で細胞を浮遊培養する工程を含む、細胞凝集塊の製造方法である。
本発明の他の一態様は、細胞と、培地と、SRF阻害剤、又はSRF阻害剤及びROCK阻害剤とを含む、細胞培養組成物である。
本発明の他の一態様は、培地と、SRF阻害剤、又はSRF阻害剤及びROCK阻害剤とを含む、細胞培養培地である。
ヒトiPS細胞として、TkDN4−M株(東京大学医科学研究所)を使用した。Vitronectin(サーモフィッシャーサイエンティフィック株式会社)をコートした細胞培養用ディッシュ上にヒトiPS細胞を播種し、培地はEssential 8TM(サーモフィッシャーサイエンティフィック株式会社)を使用して維持培養した。継代時の細胞剥離剤としては、Accutase(サーモフィッシャーサイエンティフィック株式会社)を用いた。また、細胞播種時のみ、Y−27632(和光純薬工業株式会社)を10μMの濃度になるように培地に添加した。培地交換は毎日実施した。実験には継代数50回までのヒトiPS細胞を使用した。
SRF阻害剤添加による細胞の凝集促進効果について検討した。
実施例1の手順で培養したヒトiPS細胞をAccutaseで3分間から5分間処理して剥離し、単細胞まで分散した。この細胞を最終濃度5mg/mLのBSA(和光純薬工業株式会社)および最終濃度2.5μMのY−27632(和光純薬工業株式会社)を含むEssential 8TM培地で懸濁し、その一部をトリパンブルー染色して細胞数を調べた。1mLあたり2×105個の細胞を含むように調製した。別途CCG−1423(Cayman、10010350)を最終濃度4・55mg/mL(10mM)になるように細胞凝集促進剤を調整し、前記調整した細胞凝集促進剤を前記細胞懸濁液にCCG−1423の最終濃度が10μMとなるように添加した上で、浮遊培養用12ウェルプレート(住友ベークライト株式会社)に1.3mL/ウェルの割合で播種した。細胞を播種したプレートは、ロータリーシェーカー(株式会社オプティマ)上で90 rpmのスピードで水平面に沿って旋回幅(直径)が25mmの円を描くように旋回培養し、5%CO2、37℃の環境下で浮遊培養を行った。コントロール試験として、CCG−1423を添加していない以外は上記と同様に調製した細胞懸濁液を用いた条件での試験を行った。
また、Cytotoxicity LDH Assay Kit−WST(株式会社同仁化学研究所)を用いて、死細胞数を算出した。
上記浮遊培養後(培養1日目)の顕微鏡観察像を図1に示す。観察の結果、コントロール試験(0μM CCG−1423)に比べて、CCG−1423を添加した条件では比較的大きな凝集塊を形成しており、凝集が促進されていた。死細胞数を調べた結果、コントロール試験(0μM CCG−1423)に比べて、CCG−1423を添加した条件では死細胞数が減少していた(図2)。
ヒトiPS細胞の浮遊培養を行いグルコース消費量、細胞収量、未分化マーカーの陽性率を測定し、CCG−1423が与える細胞への影響を解析した。
実施例2と同様に細胞懸濁液を調製し、別途CCG−1423(同上)を最終濃度4・55mg/mL(10mM)になるように細胞凝集促進剤を調整し、前記調整した細胞凝集促進剤を前記細胞懸濁液にCCG−1423の最終濃度が10μMとなるように添加した上で、浮遊培養用6ウェルプレート(住友ベークライト株式会社)に4mL/ウェルの割合で播種した。細胞を播種したプレートは、ロータリーシェーカー(株式会社オプティマ)上で90 rpmのスピードで水平面に沿って旋回幅(直径)が25mmの円を描くように旋回培養し、5%CO2、37℃の環境下で浮遊培養を行った。培養翌日(培養1日目)以降、毎日新鮮な培地(最終濃度5mg/mLのBSA(和光純薬工業株式会社)を含むEssential 8TM培地)に培地交換し、培養5日後まで培養を続けた。コントロール試験として、CCG−1423およびY−27632を含まない培地で同様に培養した。培養中、毎日位相差顕微鏡により画像を取得とした。培養1日目に取得した画像中の208個の細胞凝集塊を観察し、顕微鏡写真のスケールと比較して各細胞凝集塊の最も広い部分の幅(「φ」とする)を求め、その分布を調べ、平均値±標準偏差を算出した。培地交換時に回収した培養上清に含まれるグルコースの濃度を、バイオセンサーBF−5iD(王子計測機器株式会社)で測定し、グルコース消費量を計算した。
図3は培養1日目から5日目に観察した顕微鏡写真である。CCG−1423を添加した条件では播種後から培養1日目にかけて細胞凝集塊が形成されており、培養を継続することで徐々に細胞が増殖し、細胞凝集塊が大きくなった。
Claims (17)
- SRF阻害剤を含む、細胞の浮遊培養に用いるための細胞凝集促進剤。
- 前記SRF阻害剤の濃度が9.0μg/mL以上10.0mg/mL以下である、請求項1に記載の細胞凝集促進剤。
- さらにROCK阻害剤を含む、請求項1又は2に記載の細胞凝集促進剤。
- 前記SRF阻害剤がCCG−1423である、請求項1から3のいずれか1項に記載の細胞凝集促進剤。
- 前記細胞が幹細胞である、請求項1から4のいずれか1項に記載の細胞凝集促進剤。
- SRF阻害剤を含む培地中で細胞を浮遊培養する工程を含む、細胞凝集塊の製造方法。
- 前記培地中における前記SRF阻害剤の濃度が4.5ng/mL以上4.6mg/mL以下である、請求項6に記載の製造方法。
- さらに、前記培地中にROCK阻害剤を含む、請求項6又は7に記載の製造方法。
- 前記SRF阻害剤がCCG−1423である、請求項6から8のいずれか1項に記載の製造方法。
- 前記細胞が幹細胞である、請求項6から9のいずれか1項に記載の製造方法。
- 請求項6から10のいずれか1項に記載の製造方法により得られた細胞凝集塊。
- 細胞と、培地と、SRF阻害剤とを含む、細胞培養組成物。
- 前記SRF阻害剤の濃度が4.5ng/mL以上4.6mg/mL以下である、請求項12に記載の細胞培養組成物。
- さらにROCK阻害剤を含む、請求項12又は13に記載の細胞培養組成物。
- 前記SRF阻害剤がCCG−1423である、請求項12から14のいずれか1項に記載の細胞培養組成物。
- 前記細胞が幹細胞である、請求項12から15のいずれか1項に記載の細胞培養組成物。
- 前記細胞の形態が細胞凝集塊である、請求項12から16のいずれか1項に記載の細胞培養組成物。
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