JP2016521614A - 組織工学のためのマトリックス及び移植片 - Google Patents
組織工学のためのマトリックス及び移植片 Download PDFInfo
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Abstract
Description
及び
細孔形成(porogenic)浸出(leachable)材料の粒子であって、該細孔形成粒子は、ポリマー溶媒中で不溶であって、約100μm〜400μmの範囲の粒径を有する粒子、
を含む、混合物(M)を調製すること;
を含む。
e1)遠心分離;及び
e2)真空下での凍結乾燥(freeze−drying)及び/又は凍結乾燥(lyophilisation)」
を含む。
を含む。
i)本発明の少なくとも一つのマトリックス、及び
ii)培養液中で予め集合させた細胞クラスターを含む、多細胞性の微細組織スフェロイド、
を含む。
マトリックス調製
直径20mm及び厚さ2mmのマトリックスは、以下の手順によって調製された:
三次元の一次構造は、篩にかけられた(sieved)塩化ナトリウム粒子を使用した、粒子浸出技術(particulate−leaching technique)によってPGLAから調製された。
355〜425μmの直径の範囲にある、篩にかけられたNaCl粒子(36.0g)は、アルミニウム皿に置かれ、27(w/v)%濃度にてクロロホルム(17.8mL)中のPLGA溶液と収容した。このように、PLGAは、クロロホルム中で溶解され、その後、ポロゲンとしてのNaCl粒子で充填されたアルミニウム皿上にキャスト(cast)された。
マトリックスの多孔度は、以下の公式によって決定された:
親水性は、以下のように試験された:液体がマトリックスによって吸収されるか、又は、はじかれるかを決定するために、300μLのリン酸緩衝生理食塩水(PBS;シグマ社)は、液滴方法によってマトリックスの上部にピペットで移された。
マトリックス上の細胞接着は、コラーゲンタイプ1でマトリックスをコートすることによって調べられ、液滴方法によってマトリックスの上部に細胞懸濁液の300μLをピペットで移された。その後、細胞懸濁液が載置されたマトリックスは、37℃、5%CO2、及び95%相対湿度でインキュベートされた。懸濁液中に残存し、それゆえ、マトリックスに接着しなかった細胞は、媒体を変える間に、1日及び三日後にカウントされた。
細胞数は、0.2%トリパンブルー溶液で2分間、細胞懸濁系を染色した後、血球計(ノイバウアー改善チャンバー)で決定された。
−EGTA:エチレングリコール四酢酸(シグマ E4378)
−コラゲナーゼ タイプI:(ワーシントン 4196; 246U/mg)
−FCS:ウシ胎児血清(シグマ F2442)
−HEPES:4−(2−ヒドロキシエチル)−1−ピペラジンエタンスルホン酸(シグマ)
−ウィリアム メディウム E:「ウィリアムE培地(シグマ W4128)」及び10体積%のドナーの血清、又は、10体積%FCSを含む肝細胞培養用標準培地
Claims (22)
- 組織工学用多孔質マトリックスであって、一次細孔を有する三次元の一次構造を形成する、第一の生分解性及生体適合性のポリマー成分を含むと共に、コラーゲン、ラミニン、フィブロネクチン及びその混合物からなる群から選択される、該第一のポリマー成分とは異なる第二の生分解性及び生体適合性のポリマー成分をさらに含み、ここで、該第二のポリマー成分は、二次細孔を有する三次元の二次構造を形成し、該二次構造は、該一次細孔の少なくとも一部の内部空間内に含まれる、マトリックス。
- 該第一のポリマー成分が、ポリ(グリコール酸)、ポリ(乳酸)、ポリ(グリコール酸−乳酸)及びその混合物からなる群から選択される、請求項1に記載のマトリックス。
- 該第二のポリマー成分は、コラーゲン、好ましくは、ゼラチンである、請求項1又は2に記載のマトリックス。
- 該第一細孔は、150μm〜300μm、好ましくは、200μm〜300μmの平均孔径を有する、請求項1〜3のいずれか1項に記載のマトリックス。
- 該二次細孔は、該一次細孔の平均孔径より小さい平均孔径を有し、50μm〜290μm、好ましくは50μm〜150μm、より好ましくは50μm〜100μmの範囲である、請求項4に記載のマトリックス。
- 多孔質基層及び多孔質細胞包埋層を含み、基層中の細孔の平均孔径が、細胞包埋層の細孔の平均孔径よりも小さい、請求項1〜5のいずれか1項に記載のマトリックス。
- 基層の細孔は、10μm〜50μmの平均孔径を有する、請求項1〜6のいずれか1項に記載のマトリックス。
- 少なくとも80%の、好ましくは少なくとも90%の、最も好ましくは95%〜99.5%の多孔度を有する、請求項1〜7のいずれか1項に記載のマトリックス。
- 請求項1〜8のいずれか1項に記載のマトリックスを調製する方法であって、
a)ポリマー溶媒(S−I)に溶解された第一ポリマー成分のポリマー粒子のポリマー溶液(PS−I)、及び
細孔形成浸出材料の粒子、を含む混合物(M)を提供し、ここで、前記粒子は、該ポリマー溶媒(S−I)に不溶であって、約100μm〜400μmの範囲の粒径を有し、
b)該ポリマー溶媒(S−I)の除去によって該混合物を圧縮し
c)該細孔形成侵出材料を除去することにより、三次元の一次構造に一次細孔を取得し、
d)溶媒に溶解された第二ポリマー成分を含む溶液(PS−II)に一次構造を浸漬し、
e)該溶媒(S−II)を除去する、
ことを含む、方法。 - 工程a)において、該混合物(M)は、該第一のポリマー成分の固体ポリマー粒子をさらに含むと共に、該第一ポリマー成分の固体ポリマー粒子及び細孔形成材料の粒子を含む粒子混合物(Mp)にポリマー溶液(PS−I)を添加することによって調製される、請求項9に記載の方法。
- 該第一のポリマー成分の該ポリマー粒子は、約100μm〜300μmの範囲の粒径を有する、請求項9又は10に記載の方法。
- 工程d)において、該第二のポリマー成分を溶解するために使用される溶媒(S−II)が、水溶性溶媒であって、工程e)における溶媒(S−II)の除去は、
e1)遠心分離;及び、
e2)真空下でのフリーズドライ(freeze-drying)及び/又は凍結乾燥(lyophilisation)
を含む、請求項9又は11に記載の方法。 - 組織工学用移植片の製造における、特に肝臓移植片のための、請求項1〜8のいずれか1項に記載のマトリックスの使用。
- 請求項1〜8のいずれか1項に記載のマトリックス、及び、少なくとも1つのランゲルハンス島細胞を含む、組織工学用移植片。
- 肝細胞をさらに含む、請求項14に記載の移植片。
- 肝細胞対ランゲルハンス島細胞の比が、肝細胞約1×106対ランゲルハンス島細胞20,000〜500,000、好ましくは肝細胞約1×106対ランゲルハンス島細胞20,000〜100,000、最も好ましくは肝細胞約1×106対ランゲルハンス島細胞20,000〜70,000である、請求項15に記載の移植片。
- 慢性肝臓疾患若しくは膵臓疾患並びに/又は慢性肝臓障害及び膵臓障害の治療における使用のための、請求項14〜16のいずれか1項に記載の移植片。
- 請求項14〜17のいずれか1項に記載の移植片を調製するための方法であって、
a.請求項1〜8のいずれか1項に記載の多孔質マトリックスを提供する工程、及び
b.該マトリックスの少なくとも一つの側の上に、肝細胞と、少なくとも一つのランゲルハンス島細胞を載置する工程、
を含む、方法。 - 該マトリックスは実質的に、約0.1〜1.0mmの範囲の厚さを有するシートの形状であって、該方法は、さらに、
c.二層又は多層構造を得るために、細胞を載置したマトリックスを折り畳む工程、
を含む、請求項18に記載の方法。 - 工程b)が、マトリックスの両側に細胞を載置することを含む、請求項18又は19に記載の方法。
- 少なくとも一つの請求項1〜8のいずれか1項に記載のマトリックスと、予め集合させた細胞クラスターを培地中に含む多細胞性微細組織スフェロイドとを含むキット。
- 予め集合させた細胞クラスターが、少なくとも一つのランゲルハンス島細胞及び肝細胞を含む、請求項21に記載のキット。
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PCT/EP2014/001626 WO2014202199A1 (en) | 2013-06-17 | 2014-06-16 | Matrix and implant for tissue engineering |
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EP (2) | EP2815773B1 (ja) |
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WO2020105381A1 (ja) * | 2018-11-22 | 2020-05-28 | 株式会社日立製作所 | 細胞デバイス、細胞デバイスの製造方法及び細胞デバイスの移植方法 |
JP2022523952A (ja) * | 2019-03-04 | 2022-04-27 | ハンス ウー.ベーア | 外科手術後の、特にヘルニア修復における癒着を予防するための、生体分解性の二層マトリックス |
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KR102208200B1 (ko) * | 2013-08-09 | 2021-01-27 | 킴벌리-클라크 월드와이드, 인크. | 3차원 인쇄용 중합체 물질 |
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JP2022523952A (ja) * | 2019-03-04 | 2022-04-27 | ハンス ウー.ベーア | 外科手術後の、特にヘルニア修復における癒着を予防するための、生体分解性の二層マトリックス |
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CN105324136B (zh) | 2018-11-06 |
EP3010556B1 (en) | 2019-01-30 |
ES2721203T3 (es) | 2019-07-29 |
EP2815773B1 (en) | 2017-08-30 |
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