JP2023520621A - 心臓オルガノイド培養及び移植のための脱細胞心臓組織由来支持体及びその製造方法 - Google Patents
心臓オルガノイド培養及び移植のための脱細胞心臓組織由来支持体及びその製造方法 Download PDFInfo
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Abstract
Description
実施例1-1.脱細胞心臓組織由来細胞外基質の製作及び分析
図1に示すように、脱細胞工程を通じてブタ心臓組織から脱細胞心臓組織由来細胞外基質(Heart Extracellular Matrix、HEM)支持体を製作し、その特性を分析した。
凍結乾燥することで製作されたパウダー状の脱細胞心臓組織由来支持体(Lyophilized HEM)10mgを4mg/mlのペプシン溶液(ブタ胃粘膜由来ペプシンパウダー(4mg)を0.02MのHCL(1ml)に溶かした溶液)処理し、48時間常温で溶液化過程を進行した。その後、細胞培養に使用できるように10×PBS及び1MのNaOHを利用して中性のpHと1×PBSバッファの電解質状態に合わせておいた。最後に、インキュベータ内の37℃温度条件において30分間ハイドロジェルの形成(gelation)を誘導した。
実験例1-1.心臓オルガノイド培養のための脱細胞心臓組織由来細胞外基質支持体(Heart Extracellular Matrix、HEM)タンパク体の分析
脱細胞心臓組織由来支持体に含有された細胞外基質成分を確認するために、質量分析器を利用したタンパク体の分析を実施した。
HEMに含まれたmatrisomeタンパク質を、質量分析器を利用したタンパク体分析を通じて検出してタンパク質の種類別に分類し、相対的な定量分析を進行した。
本発明の脱細胞心臓組織由来細胞外基質支持体(Heart Extracellular Matrix、HEM)のnon-matrisomeタンパク質を分析した。
本発明の脱細胞方法(Protocol 1)と他の脱細胞方法(Protocol 2)とを比較するための実験を進行した。プロトコル2は、プロトコル1とは異なり、1%のsodium dodecyl sulfate(SDS)処理はせず、1%のTriton-×100及び0.1%のNH4OHを6時間だけ処理した。
脱細胞心臓組織由来HEMハイドロジェルと心筋細胞を利用して心臓オルガノイドを製作する2つの方法を図7のように行った。
実験例2-1.ヒト脱分化幹細胞由来心筋細胞の製作及びオルガノイドのカプセル化方法を利用した脱細胞心臓組織由来細胞外基質支持体(Heart Extracellular Matrix、HEM)ベースのヒト心臓オルガノイドの製作
ヒト誘導万能幹細胞を心筋細胞へ分化誘導し、分化された心筋細胞をマイクロウェル(Microwell)を利用して3次元オルガノイド形態に製作した。その後、心臓オルガノイドをHEMハイドロジェル(4mg/ml)内で3次元培養することで、分化及び成熟度が増進された心臓オルガノイドを製作した。
HEMハイドロジェル(4mg/ml)内において3次元培養したヒト心臓オルガノイドの分化増進程度を確認するために、培養7日目にqPCR分析を進行した。コラーゲンハイドロジェル(Col I)及びマトリゲル(Mat)を利用して培養したグループと、マトリックスなしに培養液に浮遊培養したグループ(No ECM)とを比較群として適用した。
HEMハイドロジェルがヒト心臓オルガノイドの心筋分化に及ぼす影響を確認するために、培養7日目に心筋特異的マーカーであるTroponin I(TNNI)に対する免疫染色を実施した。コラーゲンハイドロジェル(Col I)及びマトリゲル(Mat)を利用して培養したグループと、マトリックスなしに培養液に浮遊培養したグループ(No ECM)とを比較群として使用した。
HEMハイドロジェルが心臓オルガノイドの心筋分化に及ぼす影響を確認するために、培養7日目に心筋細胞特異的マーカーであるα-actinin及びcTnTに対する免疫染色を実施することで、当該マーカーのタンパク質発現水準を比較した。コラーゲンハイドロジェル(Col I)及びマトリゲル(Mat)を利用して培養したグループを比較群として適用した。
HEMハイドロジェル(4mg/ml)において培養したヒト心臓オルガノイドの薬物に対する反応性を確認するために、心筋拍動数に影響を及ぼす2つの代表薬物(isoproterenol及びpropranolol)を培養7日目に処理し、それに対するオルガノイドの反応をカルシウムイメージングを通じて確認した。
実験例3-1.単一心筋細胞のカプセル化方法を通じた脱細胞心臓組織由来細胞外基質支持体(Heart Extracellular Matrix、HEM)ベースのヒト心臓オルガノイドの製作方法及び遺伝子発現の分析
形成されたオルガノイドをHEMハイドロジェル内にカプセル化する方法とは異なり、ヒト誘導万能幹細胞由来の心筋細胞を単一細胞の状態でHEMハイドロジェルにカプセル化することによりヒト心臓オルガノイドを製作する方法を試みた。
オルガノイド内部の血管構造の形成は、オルガノイドの成長、発達及び機能において非常に重要な要素であるため、心臓オルガノイドの血管化のためにHEMハイドロジェル内において血管細胞が培養可能であるか否かを調べた。
構造的、機能的にさらに高度化された血管構造を備えた心臓モデルを製作するために、実際の心臓に存在する非心筋細胞(Non-myocyte)である血管細胞(endothelial cell)と心臓線維芽細胞(cardiac fibroblast)とを共にHEMハイドロジェルに単一細胞のカプセル化方式で封入することによって、3種の細胞が共培養された心臓オルガノイドを製作した。血管細胞はHUVEC細胞を利用し、心臓線維芽細胞はヒト誘導万能幹細胞から分化して使用した(図15(A))。
心筋細胞と2種の非心筋細胞(Non-myocyte)のカプセル化方法を適用した血管構造を有する高度化されたヒト心臓オルガノイドの製作及びそのための脱細胞心臓組織由来細胞外基質支持体(Heart Extracellular Matrix、HEM)の濃度を選別した。
様々な濃度のHEMハイドロジェルにおいて14日間3種の細胞が共培養された心臓オルガノイドの内部構造を確認するために、組織学分析を利用して免疫染色を進行した。
さらに高度化されたヒト心臓モデルを製作するために、人体の血流を疑似する微細な培養液の流れを提供することのできるマイクロ流体チップを利用し、チップ内においてHEMハイドロジェルを利用して心臓オルガノイドを培養した。マトリゲル(Mat)は対照群として使用した。
実験例4-1.心筋細胞と2種の非心筋細胞(Non-myocyte)のカプセル化方法と脱細胞心臓組織由来細胞外基質支持体(Heart Extracellular Matrix、HEM)を利用したLQT患者の心臓オルガノイドの製作
QT延長症候群(long QT syndrome;LQT)患者の誘導万能幹細胞から分化された心筋細胞を利用し、単一細胞のカプセル化方法を通じて心臓オルガノイドを製作した。具体的に、2mg/mlの濃度のHEMハイドロジェルを利用して進行した。心筋細胞の他に2種の非心筋細胞(血管細胞及び心臓線維芽細胞)を共にカプセル化し、類型の異なるLQT患者の幹細胞(LQT2及びLQT3)から分化された心筋細胞を利用して心臓オルガノイドを製作した。
実験例4-1のようにLQT患者の誘導万能幹細胞由来心筋細胞と血管細胞、心臓線維芽細胞を2mg/mlのHEMハイドロジェル内において共培養し、それをマイクロ流体チップ内に適用して微細な培養液の流れを提供する動的培養を通じてLQT疾患の心臓オルガノイドを製作した。
Claims (9)
- 脱細胞心臓組織由来細胞外基質(Heart Extracellular Matrix、HEM)を含む、支持体組成物。
- 前記脱細胞心臓組織由来細胞外基質は、0.01~10mg/mLで含まれる、請求項1に記載の支持体組成物。
- 前記組成物は、0.1~10Hz基準の弾性係数が1~150Paである、請求項1に記載の支持体組成物。
- (a)分離した心臓組織を脱細胞化することにより、脱細胞した心臓組織を製造するステップと、
(b)前記脱細胞した心臓組織を乾燥することにより、脱細胞心臓組織(Heart Extracellular Matrix、HEM)を製造するステップとを含む、支持体組成物製造方法。 - 前記ステップ(a)は、前記分離した心臓組織を1~10℃で12~36時間0.1~2%のSDS(sodium dodecyl sulfate)処理し、脱細胞化溶液で撹拌することで脱細胞化する、請求項4に記載の支持体組成物製造方法。
- 前記脱細胞化溶液は、0.1~5%のTriton X-100及び0.01~0.5%の水酸化アンモニウムを含む、請求項5に記載の支持体組成物製造方法。
- 前記ステップ(a)の脱細胞は、心臓組織細胞が95~99.9%除去されたものである、請求項4に記載の支持体組成物製造方法。
- 前記ステップ(b)の後、脱細胞心臓組織由来細胞外基質は、0.01~10mg/mLで含まれるように調節するステップをさらに含む、請求項4に記載の支持体組成物製造方法。
- 請求項1の支持体組成物又は請求項4の製造方法によって製造された支持体組成物で心臓オルガノイドを培養する方法。
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