JP6140240B2 - 組織工学的に作製された絹製臓器 - Google Patents
組織工学的に作製された絹製臓器 Download PDFInfo
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- JP6140240B2 JP6140240B2 JP2015204147A JP2015204147A JP6140240B2 JP 6140240 B2 JP6140240 B2 JP 6140240B2 JP 2015204147 A JP2015204147 A JP 2015204147A JP 2015204147 A JP2015204147 A JP 2015204147A JP 6140240 B2 JP6140240 B2 JP 6140240B2
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Description
本発明は、NIH TERC助成金第P41 EB002520号による援助を受けた。政府は本発明に対して一定の権利を有する。
本出願は、2007年9月17日に提出された米国仮出願第60/973,023号、および2007年2月27日に提出された米国仮出願第60/903,800号に対する優先権を主張し、これらは両方ともその全体が参照により本明細書に組み入れられる。
本発明は、組織工学的に作製された絹製角膜(tissue-engineered silk cornea)などの組織工学的に作製された絹製臓器、および組織工学的に作製された絹製臓器を製造するために用いられる方法に関する。
角膜障害は毎年、白内障に次いで、世界の一般集団における重大な視覚喪失の第2位の原因となっている。角膜置換は、急速に多くの人々にとって不可欠になりつつある開発中の技術である。角膜置換療法の理由には、疾患(例えば、ヘルペス感染)に由来する瘢痕、LASIK手術に由来する合併症、遺伝性異常(例えば、フックス病)、および他の手術(例えば、白内障)に由来する合併症といった多くのものが挙げられる。
[請求項1001]
組織特異的細胞を含む溝付き絹フィブロイン基体(grooved silk fibroin substrate)を含む、ラメラ組織層(lamellae tissue layer)。
[請求項1002]
組織特異的細胞が基体上に細胞外マトリックスを沈着させる、請求項1001記載の組織層。
[請求項1003]
溝付き絹フィブロイン基体の厚さが10nmから1mmまでの範囲であり、溝のサイズが少なくとも125nmであり、かつ溝の厚さ深さが少なくとも100nmである、請求項1001記載の組織層。
[請求項1004]
組織特異的細胞が、幹細胞、線維芽細胞、内皮細胞、上皮細胞、組織特異的細胞外マトリックスを生成することのできる脂肪細胞、およびそれらの組み合わせからなる群より選択される、請求項1001記載の組織層。
[請求項1005]
多数の請求項1001記載のラメラ組織層を含む、組織工学的に作製された臓器(tissue-engineered organ)。
[請求項1006]
各層への栄養分の拡散または脈管形成を可能にするミクロ孔(micropore)を含む、請求項1005記載の組織工学的に作製された臓器。
[請求項1007]
ミクロ孔の平均直径が500nmから100ミクロンまでの範囲である、請求項1006記載の組織工学的に作製された臓器。
[請求項1008]
ミクロ孔が、ポリ(エチレンオキシド)相分離化学法、レーザー焼灼法、またはそれらの組み合わせの使用を通じて作り出される、請求項1007記載の組織工学的に作製された臓器。
[請求項1009]
培養細胞および沈着した細胞外マトリックスを伴う絹フィブロイン基体の集成によって形成されうる任意の組織である、請求項1005記載の組織工学的に作製された臓器。
[請求項1010]
角膜である、請求項1009記載の組織工学的に作製された臓器。
[請求項1011]
光学的に透明で、非免疫原性で、かつ生体適合性である、請求項1005記載の組織工学的に作製された臓器。
[請求項1012]
組織工学的に作製された臓器を調製するための方法であって、以下の段階を含む方法:
細胞外マトリックス沈着を行うことができる組織特異的細胞が播種された溝付き絹フィブロイン基体を各層が含む、多数のラメラ組織層を調製する段階;ならびに
細胞および沈着した細胞外マトリックスに組織工学的に作製された臓器を形成させるのに十分な期間にわたって、絹フィブロイン基体上で細胞を培養する段階。
[請求項1013]
絹フィルム中にミクロ孔を作り出す段階をさらに含む、請求項1012記載の方法。
[請求項1014]
ミクロ孔が、ポリ(エチレンオキシド)相分離化学法、レーザー焼灼法、またはそれらの組み合わせの使用を通じて作り出される、請求項1013記載の方法。
[請求項1015]
ミクロ孔の平均直径が500nmから100ミクロンまでの範囲である、請求項1013記載の方法。
[請求項1016]
組織工学的に作製された臓器が非免疫原性かつ生体適合性である、請求項1012記載の方法。
[請求項1017]
臓器が、骨、皮膚、心組織、心筋、筋組織、緻密(dense)結合組織、基底膜、平滑筋組織、内皮組織、および神経組織からなる群より選択される、請求項1012記載の方法。
[請求項1018]
臓器が角膜である、請求項1017記載の方法。
[請求項1019]
組織特異的細胞が、幹細胞、線維芽細胞、組織特異的細胞外マトリックスを生成することのできる脂肪細胞、およびそれらの組み合わせからなる群より選択される、請求項1018記載の方法。
[請求項1020]
組織工学的に作製された臓器の調製が、内皮細胞シートをラメラ組織層の底面に追加し、上皮細胞シートをラメラ組織層の上面に追加する段階をさらに含む、請求項1019記載の方法。
[請求項1021]
以下のものを含む、組織工学的に作製された角膜:
細胞外マトリックス沈着物を有する線維芽細胞が播種された溝付き絹フィブロイン基体を各層が含む、少なくとも2つのラメラ組織層;
ラメラ組織層の底面上にある内皮細胞シート;および
ラメラ組織層の上面上にある上皮細胞シート。
[請求項1022]
光学的に透明で、非免疫原性で、かつ生体適合性である、請求項1021記載の組織工学的に作製された角膜。
[請求項1023]
ラメラ組織層が、各層への栄養分の拡散を可能にするミクロ孔を含む、請求項1021記載の組織工学的に作製された角膜。
[請求項1024]
以下の段階を含む、組織工学的に作製された臓器のインビボ植え込み(implantation)システム:
溝付き絹フィブロイン基体上で、培養された組織特異的細胞を含む組織工学的に作製された臓器を形成させる段階;
組織工学的に作製された臓器内の絹フィブロイン材料を少なくとも部分的に分解させる段階;および
組織工学的に作製された臓器をネイティブ(native)組織に植え込む段階。
[請求項1025]
組織工学的に作製された臓器が多数のラメラ組織層を含み、各層が溝付き絹フィブロイン基体上に培養された組織特異的細胞を含む、請求項1024記載の植え込みシステム。
[請求項1026]
組織工学的に作製された臓器が、臓器の各層への栄養分の拡散を可能にするミクロ孔を含む、請求項1024記載の植え込みシステム。
[請求項1027]
組織工学的に作製された臓器が、骨、皮膚、心組織、心筋、筋組織、緻密結合組織、基底膜、平滑筋組織、内皮組織、および神経組織からなる群より選択される、請求項1024記載の植え込みシステム。
[請求項1028]
組織工学的に作製された臓器が角膜である、請求項1024記載の植え込みシステム。
[請求項1029]
組織工学的に作製された角膜が、線維芽細胞、内皮細胞、上皮細胞、幹細胞、組織特異的細胞外マトリックスを生成することのできる脂肪細胞を含む、請求項1028記載の植え込みシステム。
[請求項1030]
組織工学的に作製された角膜が、光学的に透明で、非免疫原性で、かつ生体適合性である、請求項1028記載の植え込みシステム。
[請求項1031]
以下のものを含む、組織工学的に作製された臓器を調製するためのキット:
細胞外マトリックス沈着物を有する組織特異的細胞を受け入れることのできる溝付き絹フィブロイン基体、および
絹フィブロイン基体上に組織特異的細胞を播種するため、ラメラ組織層を形成するように絹フィブロイン基体を構成するため、絹フィブロイン基体上で組織特異的細胞を培養するため、またはそれらの組み合わせのための説明書。
[請求項1032]
初代細胞源から細胞を獲得するための収集用および格納用のツールセットをさらに含む、請求項1031記載のキット。
[請求項1033]
初代細胞源がヒトまたは動物である、請求項1032記載のキット。
[請求項1034]
絹フィブロイン基体に組織特異的細胞が播種された後に基体を培養するためのバイオリアクターをさらに含む、請求項1031記載のキット。
[請求項1035]
バイオリアクターが、組織特異的細胞が培養されるまで、無菌性で栄養分が満たされた条件を維持することができる、請求項1034記載のキット。
[請求項1036]
絹フィブロイン基体が、各層への栄養分の拡散を可能にするミクロ孔を含む、請求項1031記載のキット。
[請求項1037]
絹フィブロイン基体が非免疫原性かつ生体適合性である、請求項1031記載のキット。
[請求項1038]
組織工学的に作製された臓器を商業的検査の目的で調製するために用いられる、請求項1031記載のキット。
[請求項1039]
以下の段階を含む、動物臓器の置換のためのインビトロ検査システム:
溝付き絹フィブロイン基体上で、培養された組織特異的細胞を含む培養組織系を形成させる段階;
培養組織系を、組織特異的細胞の組織構築物がネイティブ組織の特性を維持し、かつ培養組織を形成しうるまでの期間、インキュベートする段階;および
動物臓器の置換の開発に役立てるために培養組織をインビトロ研究条件下で検査する段階。
[請求項1040]
培養組織系が多数のラメラ組織層を含み、各層が溝付き絹フィブロイン基体上に培養された組織特異的細胞を含む、請求項1039記載の検査システム。
[請求項1041]
培養組織系が、各層への栄養分の拡散を可能にするミクロ孔を含む、請求項1040記載の検査システム。
[請求項1042]
培養組織系が、骨、皮膚、心組織、心筋、筋組織、緻密結合組織、基底膜、平滑筋組織、内皮組織、および神経組織からなる群より選択される、組織工学的に作製された臓器である、請求項1041記載の検査システム。
[請求項1043]
組織工学的に作製された臓器が角膜である、請求項1042記載の検査システム。
[請求項1044]
組織工学的に作製された角膜が、幹細胞、線維芽細胞、内皮細胞、上皮細胞、組織特異的細胞外マトリックスを生成することのできる脂肪細胞、またはそれらの組み合わせを含む、請求項1043記載の検査システム。
[請求項1045]
組織工学的に作製された角膜が、光学的に透明で、非免疫原性で、かつ生体適合性である、請求項1043記載の検査システム。
[請求項1046]
絹フィブロイン基体が、1つまたは複数の生体適合性ポリマーを含む絹フィブロイン溶液から調製される、請求項1001記載のラメラ組織層。
[請求項1047]
絹フィブロイン基体が、1つまたは複数の生体適合性ポリマーを含む絹フィブロイン溶液から調製される、請求項1012記載の方法。
本発明は、組織特異的細胞を含む溝付き絹フィブロイン基体を含む、ラメラ組織層に関する。多数のラメラ組織層を用いて、ラメラ組織層を有する組織工学的に作製された臓器を作り出すことができる。
実施例1:角膜細胞増殖のための基体としての絹
絹は、多数のさまざまな細胞種に対して実用的な基体であることが示されている。絹製組織層は、Alamar Blue代謝細胞アッセイを用いた場合、組織培養用プラスチックとの比較で角膜細胞増殖を支えることもできる。図6を参照。図6に示されているように、絹、ガラスおよびTCPの基体上で同程度の細胞代謝速度が認められた。これらのデータは、ラメラ組織層を、組織工学の目的用の構成単位として製造しうることを示している。これはまた、絹製の組織工学的に作製された角膜の製造に対する裏づけも与える。
ウサギ間質線維芽細胞が絹フィブロイン基体上で増殖する能力を評価した。8%フィブロイン溶液の単層を、スピンコーター(Laurell Technologies)を用いて、12mm丸形カバーガラス上に沈着させた。スピンコーターは、ガラス表面全体にフィブロインポリマーの均等な分布を生じさせる。フィブロインでコーティングしたカバーガラスを蒸気滅菌して、24ウェル培養皿の中に置いた。初代角膜線維芽細胞を成体ウサギ角膜から単離して、TCP上で集密化するまで培養した。続いて細胞を、フィブロインでコーティングしたカバーガラス上に10,000個/cm2の密度で播種し、ダルベッコ変法イーグル培地(DMEM)、10%ウシ胎仔血清(FBS)および1%のPenstrep(細胞培養物の汚染を軽減するための抗生物質処理)を含む培地を補充した。細胞を集密化するまで増殖させたところ、細胞の形態はTCP上で増殖させた線維芽細胞と類似していた。図7を参照。
絹フィブロイン基体は細胞およびECMの発生を手引きすることのできるラメラ組織層に変容させることができ、また、栄養分の拡散を支えるために脈管形成させること、もしくは多孔性ネットワークを生成させることもできる。続いて、これらのラメラ組織層を組み合わせて、3-D組織構築物または組織工学的に作製された臓器を形成させることができる。
材料:8%絹溶液、5%ポリ(エチレンオキシド)(PEO)溶液、水およびメタノールを用いた。乾燥器、ポリジメチルスルホキシド(PDMS)基体、Teflonカバーグラス、Teflon乾燥表面、試験管立て、ピンセット、1mLシリンジ、微量遠心管および24ウェルプレート。
Claims (9)
- 組織工学的に作製された植え込み可能な臓器を調製するための方法であって、以下の段階を含む方法:
水なましにより誘導されたβ-シート結晶化を有し且つ細胞外マトリックス沈着を行うことができる組織特異的細胞が播種された、溝付き絹フィブロイン基体を各層が含む、多数のラメラ組織層を調製する段階;ならびに
細胞および沈着した細胞外マトリックスに組織工学的に作製された植え込み可能な臓器を形成させるのに十分な期間にわたって、絹フィブロイン基体上で細胞を培養する段階。 - 絹フィルム中に、各層への栄養分の拡散または脈管形成を可能にするミクロ孔を作り出す段階をさらに含む、請求項1記載の方法。
- 前記ミクロ孔が、ポリ(エチレンオキシド)相分離化学法、レーザー焼灼法、またはそれらの組み合わせの使用を通じて作り出される、請求項2記載の方法。
- 前記ミクロ孔が、500nmから100μmまでの範囲の平均直径を有する、請求項2記載の方法。
- 前記組織工学的に作製された植え込み可能な臓器が非免疫原性かつ生体適合性である、請求項1記載の方法。
- 前記組織工学的に作製された植え込み可能な臓器が、骨、皮膚、心組織、心筋、筋組織、緻密(dense)結合組織、基底膜、平滑筋組織、内皮組織、および神経組織からなる群より選択される、請求項1記載の方法。
- 前記組織工学的に作製された植え込み可能な臓器が角膜である、請求項6記載の方法。
- 前記組織特異的細胞が、幹細胞、線維芽細胞、組織特異的細胞外マトリックスを生成することのできる脂肪細胞、およびそれらの組み合わせからなる群より選択される、請求項7記載の方法。
- 前記組織工学的に作製された植え込み可能な臓器の調製が、内皮細胞シートをラメラ組織層の底面に追加し、上皮細胞シートをラメラ組織層の上面に追加する段階をさらに含む、請求項8記載の方法。
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