JPWO2018225705A1 - 細胞培養物の製造方法 - Google Patents
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Abstract
Description
<1>多能性幹細胞から分化誘導された目的細胞を含む細胞培養物を製造する方法であって、前記多能性幹細胞から前記目的細胞へと分化誘導して得られた胚様体を分散させた、前記目的細胞および該目的細胞に対して相対的に増殖速度の早い細胞を含む細胞集団を、コンフルエントに達する密度またはそれ以上の密度で播種することを含む、前記方法。
<2>コンフルエントに達する密度が、接触阻害により細胞の増殖を実質的に停止する密度である、上記<1>に記載の方法。
<3>細胞培養物が、目的細胞を、播種前の細胞集団よりも高い割合で含有する細胞培養物である、上記<1>または<2>に記載の方法。
<4>細胞培養物が、目的細胞に対して相対的に増殖速度の早い細胞群を、播種前の細胞集団よりも低い割合で含有する細胞培養物である、上記<1>〜<3>のいずれか一つに記載の方法。
<5>腫瘍形成能を有する細胞を除去することをさらに含む、上記<1>〜<4>のいずれか一つに記載の方法。
<6>腫瘍形成能を有する細胞を除去することが、ブレンツキシマブ・ベドチンを用いて処理することを含む、上記<5>に記載の方法。
<7>多能性幹細胞が、iPS細胞である、上記<1>〜<6>のいずれか一つに記載の方法。
<8>多能性幹細胞が、ヒト細胞である、上記<1>〜<7>のいずれか一つに記載の方法。
<9>目的細胞が、それを必要とする対象に適用するための細胞である、上記<1>〜<8>のいずれか一つに記載の方法。
<10>目的細胞が、心臓、肺、肝臓、膵臓、腎臓、大腸、小腸、脊髄、中枢神経系、骨、眼、皮膚、血管または血液に適用される、上記<9>に記載の方法。
<11>目的細胞が、間葉系幹細胞、骨格筋芽細胞、多分化性心臓前駆細胞、単能性心臓前駆細胞、心筋細胞、骨格筋細胞、平滑筋細胞、血管芽細胞、上皮細胞、内皮細胞、肺細胞、肝細胞、膵細胞、腎細胞、副腎細胞、腸管上皮細胞、神経幹細胞、骨髄間質細胞、神経細胞、角膜上皮細胞、角膜内皮細胞、網膜色素上皮細胞、T細胞、NK細胞、NKT細胞、樹状細胞、または血球細胞である、上記<1>〜<10>のいずれか一つに記載の方法。
<12>目的細胞が、心筋細胞である、上記<1>〜<10>のいずれか一つに記載の方法。
<13>トロポニン陽性率が、50%〜90%である細胞培養物が得られる、上記<1>〜<12>のいずれか一つに記載の方法。
<14>Lin28陽性率が、0.30%以下である細胞培養物が得られる、上記<1>〜<13>のいずれか一つに記載の方法。
<15>シート状細胞培養物を製造する方法であって、上記<1>〜<14>のいずれか一つに記載の方法により得られた細胞培養物をシート化することを含む、前記方法。
本明細書において別様に定義されない限り、本明細書で用いる全ての技術用語および科学用語は、当業者が通常理解しているものと同じ意味を有する。本明細書中で参照する全ての特許、出願および他の出版物や情報は、その全体を参照により本明細書に援用する。また本明細書において参照された出版物と本明細書の記載に矛盾が生じた場合は、本明細書の記載が優先されるものとする。
(1)樹立されたヒトiPS細胞を、フィーダー細胞を含まない培養液で維持培養するステップ(フィーダーフリー法)、
(2)得られたiPS細胞から胚様体を形成するステップ、
(3)得られた胚様体をアクチビンA、骨形成タンパク質(BMP)4および塩基性線維芽細胞増殖因子(bFGF)を含有する培養液中で培養するステップ、
(4)得られた胚様体をWnt阻害剤、BMP4阻害剤およびTGFβ阻害剤を含む培養液中で培養するステップ、および
(5)得られた胚様体をVEGFおよびbFGFを含む培養液中で培養するステップ
を含む、方法が挙げられる。
本発明において、胚様体の分散は、既知の任意の手法を用いて行うことができる。かかる手法としては、限定されずに、例えば、トリプシン/EDTA、プロナーゼ、ディスパーゼ、コラゲナーゼ、CTK(リプロセル)、TrypLE(登録商標)Select(Thermo Fisher Scientific)などを細胞分散剤として用いて分散する化学的な方法、ピペッティングなどによる物理的方法などが挙げられる。
本発明の一態様において、得られる細胞培養物中の細胞のトロポニン陽性率は、60%〜80%であり、かつ、Lin28陽性率は、0.30%〜0.20%である。
本発明の一態様において、得られる細胞培養物中の細胞のLin28陽性率の範囲は、例えば、0.35%〜0.10%、0.30%〜0.20%、0.25%〜0.20%などであり得る。
(1)ヒトiPS細胞の維持培養・分化誘導
臨床用ヒトiPS細胞株は京都大学Ciraで樹立されたものを用い、Nakagawa M. et al., Scientific Reports, 4:3594 (2014)を参考に、フィーダーフリー法で維持培養した。分化誘導法はMiki K. Cell Stem Cell (2015)、WO2014/185358A1およびWO2017/038562を参考に実施した。
具体的には、フィーダー細胞を含まない培養液で維持培養したヒトiPS細胞を、EZ Sphere(旭硝子)上で10μM Y27632(和光純薬)を含有するStemFit AK03培地(味の素)中で1日培養し、得られた胚様体をアクチビンA、骨形成タンパク質(BMP)4および塩基性線維芽細胞増殖因子(bFGF)を含有する培養液中で培養し、さらにWnt阻害剤(IWP3)およびBMP4阻害剤(Dorsomorphin)およびTGFβ阻害剤(SB431542)を含む培養液中で培養し、その後VEGFおよびbFGFを含む培養液中で培養を行った。
分化誘導後の心筋細胞を含む胚様体に対して、TrypLE(登録商標)Select Enzyme (10X),no phenol red(Thermo Fisher Scientific)を1mM EDTAにて3×の濃度に希釈した溶液を用い、37℃で10分間インキュベートすることにより、単一細胞へと分散した。残存する細胞凝集物をストレイナー(BD Bioscience)で除去し、その後の実験に供した。
ゼラチンコートした6wellプレート(培養面積9.6cm2)に、DMEM High Glucose培地(ナカライテスク,08458-16)にFBS(Moregate, 553-04423)を10%含む培地(以下、DMEM-10%FBS培地)に10μMのY27632(ROCK阻害剤)を入れ、分散させた心筋細胞を、1.8×107個播種した。アドセトリス(登録商標)処理を行う場合、その翌日よりアドセトリス(登録商標)処理を行ない、5μg/ml、48時間の処理をした。その後、DMEM-10%FBS培地に培地交換して、48時間培養を継続した。
トリパンブルー染色を行うことにより、細胞数を算出し、播種細胞数に対する回収した生細胞数から細胞回収率を算出した。心筋細胞純度は、分散した細胞をBD Cytofix/Cytoperm(登録商標)Fixation/Permeabilization Solution Kit(BD Bioscience)を用いて固定、透過処理した後、抗ヒトトロポニン抗体(Thermo Fisher Scientific)、標識2次抗体(Thermo Fisher Scientific)を順次反応させた後、フローサイトメーターにより測定を行った。未分化細胞マーカーであるLin28を発現する細胞数の割合は定量PCRで測定した。
結果を図1に示す。播種前の細胞集団のトロポニン陽性率は30%であったが、アドセトリス(登録商標)処理を行なわなかったものについては、合計培養日数5日後、播種後のトロポニン陽性率が64%となり、回収細胞数は7.7×106個で、細胞回収率は約43%、未分化細胞マーカーであるLin28陽性率は0.4%であった。
アドセトリス(登録商標)処理を行ったものについては、合計培養日数5日後、トロポニン陽性率は62%となり、回収細胞数は9.9×106個で、回収率は約56%、Lin28陽性率は0.2%であった。
同様に行った他のロットにおける結果を表1に示す。いずれのロットにおいても、合計培養日数5日後、トロポニン陽性率は、播種前の値と比較して増加した。
本方法は、トロポニン陽性率が上昇した細胞培養物を高い回収率で得ることができるという、驚くべき、予想外の結果を示すものであった。
以下の比較例においては、WO2017/010544に記載の分化誘導方法により作製した心筋細胞を用いて実施した。
ヒトiPS細胞株253G1を理化学研究所から購入して使用した。Matsuura K. et al., Biochem Biophys Res Commun, 2012 Aug 24;425(2):321-7に記載の方法に従いリアクターを用いて心筋分化誘導を行った。具体的には、PrimateES培地(リプロセル)に5ng/mLのbFGFを添加したものを未分化維持培地として用い、マイトマイシンC処理を行ったMEF上で未分化253G1細胞を培養した。10cm培養皿10枚分の未分化253G1細胞を、剥離液(リプロセル)を用いて回収し、10μMのY27632(ROCK阻害剤)を添加したmTeSR培地(ステムセルテクノロジーズ)100mLに懸濁した後、ベッセルに移し、バイオリアクター(エイブル)で撹件培養を開始した。1日後、培地からY27632を除いた。1〜3日後に培地をStemPro-34(ライフテクノロジーズ)置換し、3日〜4日後に0.5ng/mLのBMP4を添加し、4〜7日後に10ng/mLのBMP4と5ng/mLのbFGFと3ng/mLのアクチビンAを添加し、7〜9日後に4μMのIWR−1を添加し、9日目以降は5ng/mLのVEGFと10ng/mLのbFGFを添加して撹絆を続け、16〜18日後に細胞を回収した。こうしてヒトiPS細胞由来の心筋細胞を含む細胞集団(細胞塊)を得た。当該細胞集団は、0.05%トリプシン/EDTAで分散後、残存する細胞凝集物をストレイナー(BD Bioscience)で除去し、その後の実験に供した。
WO2017/010544、WO2007/088874およびTohyama S. et al., Cell Stem Cell 12, 127-137, Jan 3, 2013を参考に心筋細胞の精製法を比較検討した。iPS細胞由来心筋細胞をNormalGlucose培地に懸濁し、10μMのY27632(ROCK阻害剤)を入れて、回転振とう機で振とうさせて細胞凝集塊を作製した。次の日からDMEM Glucose free培地(Gibco, 11966-025)に乳酸(和光純薬工業, 129-02666)を4mMになるように添加した乳酸培地で培地交換して、合計5日間浮遊培養を継続した。その間、培養3日目に再度乳酸培地で培地交換した。培養5日目に細胞を回収し、生細胞数を測定し、トロポニン陽性率をフローサイトメーターにより測定した。結果を図3に示す。乳酸培地で精製処理する前は、トロポニン陽性率が平均26%、細胞数の平均が6.2×107個であったものが、5日後にはトロポニン陽性率が平均48%で、回収細胞数は3.0×106個となった。細胞回収率は5%であった。この方法は、トロポニン陽性率を上昇させるものの、細胞回収率を著しく低下させるものであった。
Dubois, N. C. et al., Nat. Biotechnol. 29,1011-8 (2011)を参考に抗体を用いた心筋細胞の精製法を比較検討した。iPS細胞由来心筋細胞をトロポニン陽性率が平均51%、平均1.4×107個をCD172−PE(ミルテニー, 130-099-783)に4℃で10分間反応させて、洗浄後にAnti-PE Beadsに懸濁し、4℃で15分間反応後に洗浄した。再度、洗浄液で懸濁し、磁気分離LS−カラムに通して、回収した細胞数を測定し、フローサイトメトリーでトロポニン陽性率を測定した。結果を図4に示す。回収した細胞数は、平均2.2×106個で、トロポニン陽性率は平均92%となり、細胞回収率は16%であった。この方法は、トロポニン陽性率を上昇させるものの、細胞回収率を著しく低下させるものであった。
Claims (15)
- 多能性幹細胞から分化誘導された目的細胞を含む細胞培養物を製造する方法であって、前記多能性幹細胞から前記目的細胞へと分化誘導して得られた胚様体を分散させた、前記目的細胞および該目的細胞に対して相対的に増殖速度の早い細胞を含む細胞集団を、コンフルエントに達する密度またはそれ以上の密度で播種することを含む、前記方法。
- コンフルエントに達する密度が、接触阻害により細胞の増殖を実質的に停止する密度である、請求項1に記載の方法。
- 細胞培養物が、目的細胞を、播種前の細胞集団よりも高い割合で含有する細胞培養物である、請求項1または2に記載の方法。
- 細胞培養物が、目的細胞に対して相対的に増殖速度の早い細胞群を、播種前の細胞集団よりも低い割合で含有する細胞培養物である、請求項1〜3のいずれか一項に記載の方法。
- 腫瘍形成能を有する細胞を除去することをさらに含む、請求項1〜4のいずれか一項に記載の方法。
- 腫瘍形成能を有する細胞を除去することが、ブレンツキシマブ・ベドチンを用いて処理することを含む、請求項5に記載の方法。
- 多能性幹細胞が、iPS細胞である、請求項1〜6のいずれか一項に記載の方法。
- 多能性幹細胞が、ヒト細胞である、請求項1〜7のいずれか一項に記載の方法。
- 目的細胞が、それを必要とする対象に適用するための細胞である、請求項1〜8のいずれか一項に記載の方法。
- 目的細胞が、心臓、肺、肝臓、膵臓、腎臓、大腸、小腸、脊髄、中枢神経系、骨、眼、皮膚、血管または血液に適用される、請求項9に記載の方法。
- 目的細胞が、間葉系幹細胞、骨格筋芽細胞、多分化性心臓前駆細胞、単能性心臓前駆細胞、心筋細胞、骨格筋細胞、平滑筋細胞、血管芽細胞、上皮細胞、内皮細胞、肺細胞、肝細胞、膵細胞、腎細胞、副腎細胞、腸管上皮細胞、神経幹細胞、骨髄間質細胞、神経細胞、角膜上皮細胞、角膜内皮細胞、網膜色素上皮細胞、T細胞、NK細胞、NKT細胞、樹状細胞、または血球細胞である、請求項1〜10のいずれか一項に記載の方法。
- 目的細胞が、心筋細胞である、請求項1〜10のいずれか一項に記載の方法。
- トロポニン陽性率が、50%〜90%である細胞培養物が得られる、請求項1〜12のいずれか一項に記載の方法。
- Lin28陽性率が、0.30%以下である細胞培養物が得られる、請求項1〜13のいずれか一項に記載の方法。
- シート状細胞培養物を製造する方法であって、請求項1〜14のいずれか一項に記載の方法により得られた細胞培養物をシート化することを含む、前記方法。
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