JP7422665B2 - 生体模倣オルガノイド培養物 - Google Patents
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Description
本発明はまた、以下に関する。
[項目1]
底部に半透膜(33)を備えたバイオリアクター容器(30)内でオルガノイド組織(10)の生体模倣共培養をする方法であって、
(a)少なくとも1種の第1の細胞種の細胞を膜(33)上に播種するステップと、
(b)前記播種細胞を培養して、前記膜(33)に支持された少なくとも1層の2D細胞層(20)を形成させるステップと、
(c)バイオリアクター容器(30)内の前記支持2D細胞層(20)上へ、
- 互いに対して規定の3D構造で配置されている少なくとも2種の更なる細胞種の細胞を含むオルガノイド(10)、及び
- ヒドロゲル(15)
を導入するステップであって、バイオリアクター容器(30)内のオルガノイド(10)が、導入されたヒドロゲル(15)を介して、前記支持2D細胞層(20)から離れて位置することを条件とするステップと
を含む、方法。
[項目2]
ステップ(c)において、バイオリアクター容器(30)内のオルガノイド(10)が、導入されたヒドロゲル(15)を介して、バイオリアクター容器(30)の壁部(38)からも離れていることを更なる条件とする、項目1に記載の方法。
[項目3]
ステップ(c)において、オルガノイド(10)がヒドロゲル(15)と共にバイオリアクター容器(30)内に導入される、項目1又は2に記載の方法。
[項目4]
ステップ(c)が、
(c1)ヒドロゲルの一部を導入して、2D細胞層(20)に対して、及び場合によりバイオリアクター容器(30)の壁部(38)に対して規定の間隔層(18)を形成させるサブステップと、次いで
(c2)形成されたヒドロゲル間隔層(18)上にオルガノイド(10)を導入するサブステップとを含む、項目1又は2に記載の方法。
[項目5]
底部の半透膜(33)上の2D細胞層(20)の培養において、その基底極(24)がその頂端極(22)と別に潅流される、項目1~4のいずれか一項に記載の方法。
[項目6]
底部に半透膜(33)を備えたバイオリアクター容器(30)内のin vitro組織培養物であって、
- 半透膜(33)上の少なくとも第1の細胞種を含む2D細胞層(20)と、
- 互いに対して規定の3D構造で配置されている少なくとも2種の更なる細胞種の細胞を含むオルガノイド(10)と、
- オルガノイド(10)がバイオリアクター容器(30)内で埋設されており、底部の2D細胞層(20)から規定の距離離れて位置している、ヒドロゲル(15)と
を含む、in vitro組織培養物。
[項目7]
オルガノイド(10)の2D細胞層(20)に対する規定の距離が、1μm~100μm、好ましくは2μm~20μmである、項目6に記載のin vitro組織培養物。
[項目8]
オルガノイド(10)が、バイオリアクター容器(30)の壁部(38)からも離れているように、ヒドロゲル(15)中に埋設されている、項目6又は7に記載のin vitro組織培養物。
[項目9]
第1の2D細胞層(20)が、膜(33)の、オルガノイド(10)に向いた上面に配置されている、項目6~8のいずれか一項に記載のin vitro組織培養物。
[項目10]
更なる2D細胞層(25)が、膜(33)の、オルガノイド(10)と反対側の下面に配置されている、項目9に記載のin vitro組織培養物。
[項目11]
2D細胞層(20、25)の細胞種が、
- 上皮細胞、
- 上皮様細胞、
- 内皮細胞、
- 線維細胞及び/又は線維芽細胞を含む間質細胞、並びに
- 筋芽細胞、筋細胞、及び/又は筋線維を含む筋肉細胞
から選択される、項目6~10のいずれか一項に記載のin vitro組織培養物。
[項目12]
膜(33)の、オルガノイド(10)に向いた上面にある第1の2D細胞層(20)が、上皮細胞を含む又は上皮細胞からなる、項目9~11のいずれか一項に記載のin vitro組織培養物。
[項目13]
膜(33)の、オルガノイド(10)と反対側の下面にある更なる2D細胞層(25)が、内皮細胞を含む又は内皮細胞からなる、項目12に記載のin vitro組織培養物。
[項目14]
オルガノイド(10)が、網膜オルガノイド、脳オルガノイド、膵臓オルガノイド、及び腸オルガノイドを含む、自己組織化多細胞種組織、又は細胞プリントによって製造可能な規定の3D構造を有する多細胞種組織の群から選択される、項目6~13のいずれか一項に記載のin vitro組織培養物。
[項目15]
オルガノイド(10)が、少なくとも光受容体細胞、及び脊椎動物の神経網膜の少なくとも1種の更なる細胞種の細胞を含む網膜オルガノイドであり、かつ2D細胞層(20)が、網膜色素上皮細胞由来のコンフルエントな単層である、項目6~14のいずれか一項に記載のin vitro組織培養物。
1.網膜MPSの製造/集成
バイオリアクターを製造するために、MPSの複数の層を、微細構造化されたシリコンウエハー上にポリジメチルシロキサン(PDMS)をキャスティングすることにより製造する。しかしながら、MPSの製造はこの材料に限定されず、ガラス、PC並びにPET及びそれらの組み合わせ物等の他の材料も可能である。それぞれの流し込み型(マスター)の微細構造化はフォトレジスト(SU-8、MicroChem社)のUVリソグラフィーによって実現される。
初めに、アセンブルされたMPSを50ワットの出力及び0.1Nml/分~0.3Nml/分の酸素ガス流量並びに5分~15分の処理期間で酸素プラズマによって滅菌する。MPSの滅菌は、同様にオートクレーブ処理又はガンマ線照射によって行うこともできる。プラズマ処理の実施後に、RPE細胞の後の接着を可能にするために半透膜の被覆を行う。このために、DMEM/F12とラミニンとの1:10~1:25希釈を適用し、MPSを37℃及び5%のCO2で1時間~4時間の期間にわたりインキュベートする(図3A)。iPS-RPE細胞をプレーティングする前に、余分なラミニン混合物を除去し、MPS全体を培地ですすぐ。既に事前に引き離されたiPS-RPE細胞を、5μl~10μlの容量で直接的に上からRO及びRPE組織チャンバーを通じて膜上に加える。iPS RPE細胞を膜上に接着させることを可能にするために、iPS RPE細胞を37℃及び5%のCO2で30分間~60分間の期間にわたりインキュベートする(図3B)。引き続き、MPS内のiPS RPE細胞を、1日間~3日間の期間にわたり外部のシリンジポンプを介して10μl/時間~20μl/時間の一定の培地流量で培養した。
MPSにおけるiPS-RO及びiPS-RPEの共培養の生理的機能性及び活力は、以下のように検出することができた:
Claims (11)
- 底部に半透膜(33)を備えたバイオリアクター容器(30)を含むバイオリアクターシステム内でオルガノイド組織(10)の生体模倣共培養をする方法であって、
(a)少なくとも1種の第1の細胞種の細胞を膜(33)上に播種するステップと、
(b)前記播種細胞を培養して、前記膜(33)に支持された少なくとも1層のコンフルエントな2D細胞層(20)を形成させるステップと、
(c)バイオリアクター容器(30)内の前記支持されたコンフルエントな2D細胞層(20)上へ、
- 互いに対して規定の3D構造で配置されている少なくとも2種の更なる細胞種の細胞を含むオルガノイド(10)、及び
- ヒドロゲル(15)
を導入するステップであって、バイオリアクター容器(30)内のオルガノイド(10)が、導入されたヒドロゲル(15)を介して、前記支持されたコンフルエントな2D細胞層(20)から離れて位置することを条件とするステップと
を含み、
コンフルエントな2D細胞層(20)の頂端極(22)は、オルガノイド(10)の方向を向いており、かつコンフルエントな2D細胞層(20)の基底極(24)は、膜(33)に向けて反対方向を向いており、
基底潅流チャネル(34)が、バイオリアクターシステムにおいて提供されており、かつコンフルエントな2D細胞層(20)の基底極(24)の潅流を可能とし、かつ
オルガノイド(10)は、少なくとも光受容体細胞、及び脊椎動物の神経網膜の少なくとも1種の更なる細胞種の細胞を含む網膜オルガノイドであり、かつコンフルエントな2D細胞層(20)が、網膜色素上皮細胞の単層である、方法。 - ステップ(c)において、バイオリアクター容器(30)内のオルガノイド(10)が、導入されたヒドロゲル(15)を介して、バイオリアクター容器(30)の壁部(38)からも離れていることを更なる条件とする、請求項1に記載の方法。
- ステップ(c)において、オルガノイド(10)がヒドロゲル(15)と共にバイオリアクター容器(30)内に導入される、請求項1又は2に記載の方法。
- ステップ(c)が、
(c1)ヒドロゲルの一部を導入して、コンフルエントな2D細胞層(20)に対して、及び場合によりバイオリアクター容器(30)の壁部(38)に対して規定の間隔層(18)を形成させるサブステップと、次いで
(c2)形成されたヒドロゲル間隔層(18)上にオルガノイド(10)を導入するサブステップとを含む、請求項1又は2に記載の方法。 - 底部の半透膜(33)上のコンフルエントな2D細胞層(20)の培養において、その基底極(24)がその頂端極(22)と別に潅流される、請求項1~4のいずれか一項に記載の方法。
- 底部に半透膜(33)を備えたバイオリアクター容器(30)を含むバイオリアクターシステム内のin vitro組織培養物であって、
- 半透膜(33)上の少なくとも第1の細胞種を含むコンフルエントな2D細胞層(20)と、
- 互いに対して規定の3D構造で配置されている少なくとも2種の更なる細胞種の細胞を含むオルガノイド(10)と、
- オルガノイド(10)がバイオリアクター容器(30)内で埋設されており、コンフルエントな底部の2D細胞層(20)から規定の距離離れて位置している、ヒドロゲル(15)と
を含み、
コンフルエントな2D細胞層(20)の頂端極(22)は、オルガノイド(10)の方向を向いており、かつコンフルエントな2D細胞層(20)の基底極(24)は、膜(33)に向けて反対方向を向いており、
基底潅流チャネル(34)が、バイオリアクターシステムにおいて提供されており、かつコンフルエントな2D細胞層(20)の基底極(24)の潅流を可能とし、かつ
オルガノイド(10)は、少なくとも光受容体細胞、及び脊椎動物の神経網膜の少なくとも1種の更なる細胞種の細胞を含む網膜オルガノイドであり、かつコンフルエントな2D細胞層(20)が、網膜色素上皮細胞の単層である、in vitro組織培養物。 - オルガノイド(10)のコンフルエントな2D細胞層(20)に対する規定の距離が、1μm~100μm、好ましくは2μm~20μmである、請求項6に記載のin vitro組織培養物。
- オルガノイド(10)が、バイオリアクター容器(30)の壁部(38)からも離れているように、ヒドロゲル(15)中に埋設されている、請求項6又は7に記載のin vitro組織培養物。
- 第1の2D細胞層(20)が、膜(33)の、オルガノイド(10)に向いた上面に配置されている、請求項6~8のいずれか一項に記載のin vitro組織培養物。
- 更なる2D細胞層(25)が、膜(33)の、オルガノイド(10)と反対側の下面に配置されている、請求項9に記載のin vitro組織培養物。
- 膜(33)の、オルガノイド(10)と反対側の下面にある更なる2D細胞層(25)が、内皮細胞を含む又は内皮細胞からなる、請求項10に記載のin vitro組織培養物。
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