JP2021531924A - 細胞受容足場を作製するための材料および方法 - Google Patents
細胞受容足場を作製するための材料および方法 Download PDFInfo
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Abstract
Description
本出願は、2018年8月2日出願の米国仮特許出願第62/713,817号の利益を主張するものである。
印刷可能な組成物の製剤.
例示的な印刷可能な組成物を、製剤A〜Fに従って調製した。表1に、各製剤に含まれるコラーゲン、モノマー、光重合開始剤、およびビヒクルの量を提供する。N−ヒドロキシエチルアクリルアミド(HEAA)、ポリエチレングリコールジメタクリレート(PEGDMA 600)、およびタートラジンは、Aldrich Corporationから購入した。メタクリレート官能化ウシコラーゲンはABM Corporationから購入した。トランスジェニックタバコ由来のヒト組換えコラーゲンは、CollPlant Ltdから購入した。ジフェニル(2,4,6−トリメチルベンゾイル)ホスフィンオキシド光重合開始剤は、製品名Omnirad TPO−LでIGN Resin Corporationから入手した。全ての重量パーセントは、インク製剤の総重量と比較した乾燥重量に基づいて決定されている。
を組み合わせることによって調製した。製剤Aのうちの約60wt%は、20mM酢酸溶液中のメタクリレート官能化コラーゲンであり、これは、約1重量%のコラーゲンである。したがって、製剤Aは、乾燥重量で約0.6%のコラーゲンを含む。製剤Aは、約10〜15重量%のHEAAモノマーを含む。製剤Aは、高グリーンストレングスが提供される一方、コラーゲン成分がなおも大きいという利点を有する。印刷可能な組成物として使用した場合、製剤Aによって、1〜50kPaの範囲のグリーンストレングスを呈する1〜15cmの三次元物品が得られた。
細胞生存率の評価.
ヒト上皮性肺癌細胞(A549、ATCC、CCL−185)を使用して、細胞生存率を評価した。細胞は、10%FBS、ペニシリン50μg mL−1、ストレプトマイシン50μg mL−1を補充したF−12K培地(Kaighnの改変、ATCC)で維持した。製剤BおよびCについて上記の通りに製剤を調製し、紫外線オーブンを使用して96ウェルプレートで硬化させた。各ウェル毎に6000個の細胞を播種し、5%CO2中37℃でインキュベートした。示されているプリント製剤それぞれで48時間インキュベートした後、細胞をPBSで3回穏やかに洗浄し、製造元の取扱説明書(Life Technologies、Thermo Fisher Scientific)に従ってPicoGreenアッセイを使用して細胞毒性評価を行った。製剤Cは、細胞播種の48時間後で80%の細胞生存率を呈した(図3)。
三次元細胞受容足場の調製.
製剤Hによる酸性の印刷可能な組成物を使用して、三次元細胞受容足場を調製した。製剤Hは、実施例1に記載の調製方法および下の表2に記載の調製方法に従って調製した。
Claims (39)
- 細胞受容足場を製造するための印刷可能な組成物であって:
前記印刷可能な組成物の重量に対して、
約0.3wt%〜約3.0wt%の1種または複数のコラーゲン;
約5.0wt%〜約40.0wt%の1種または複数のモノマー;
約0.5wt%〜約2.0wt%の光重合開始剤;および
0wt%〜約75wt%の、プロトン性溶媒を含むビヒクル
を含み、
印刷時に約100ミクロン以下の解像度、印刷時に約0.1〜5mm(Dp)および約10〜100mJ/cm2(Ec)のフォトスピード(Dp/Ec)、ならびに乾燥後に少なくとも約5kPaのグリーンストレングスを有する、印刷可能な組成物。 - 前記コラーゲンが、モノマーサブユニットで官能化されている、請求項1に記載の印刷可能な組成物。
- 前記モノマーが、アクリレート、メタクリレート、カルボキシエチルアクリレート、またはそれらのうちの2種以上の混合物からなる群から選択されるアクリルモノマーである、請求項2に記載の印刷可能な組成物。
- 前記コラーゲンが、哺乳動物源から得られる、請求項1〜3のいずれか一項に記載の印刷可能な組成物。
- 前記哺乳動物源が、ヒト、ウシ、ブタ、およびそれらのうちの2種以上の混合物からなる群から選択される、請求項4に記載の印刷可能な組成物。
- 前記コラーゲンが、組換えコラーゲンである、請求項4に記載の印刷可能な組成物。
- 前記組換えコラーゲンが、組換えヒトコラーゲン、組換えウシコラーゲン、組換えブタコラーゲン、またはそれらのうちの2種以上の混合物である、請求項6に記載の印刷可能な組成物。
- 前記組換えコラーゲンが、植物体、細菌、ウイルス、真菌、およびそれらのうちの2つ以上の組合せからなる群から選択される組換え源から得られる、請求項7に記載の印刷可能な組成物。
- 前記コラーゲンが、I型コラーゲンを含む、請求項1〜8のいずれか一項に記載の印刷可能な組成物。
- 前記コラーゲンが、印刷可能な組成物の約0.3重量パーセントから約0.9重量パーセントまでの量で存在し、前記コラーゲンは哺乳動物源由来のものである、請求項1〜9のいずれか一項に記載の印刷可能な組成物。
- 前記コラーゲンが、印刷可能な組成物の約0.5重量パーセントから約3.0重量パーセントまでの量で存在し、前記コラーゲンは組換えコラーゲン由来のものである、請求項1〜9のいずれか一項に記載の印刷可能な組成物。
- 前記コラーゲンが、第1のコラーゲンと第2のコラーゲンとの混合物であり、前記第1のコラーゲンは官能化コラーゲンであり、前記第2のコラーゲンは非官能化コラーゲンである、請求項1〜11のいずれか一項に記載の印刷可能な組成物。
- 前記第1のコラーゲンおよび前記第2のコラーゲンが、コラーゲンの乾燥重量に基づいて約99:1〜約1:99の重量比で存在する、請求項12に記載の印刷可能な組成物。
- 前記モノマーが、N−ヒドロキシエチルアクリルアミド(HEAA)、2−ヒドロキシエチルメタクリレート、ポリ(エチレングリコール)ジアクリレート、ポリエチレングリコールジメタクリレート、ポリカプロラクトンジメタクリレート、およびポリカプロラクトントリメタクリレートのうちの1種または複数を含む、請求項1〜13のいずれか一項に記載の印刷可能な組成物。
- 前記モノマーが、N−ヒドロキシエチルアクリルアミド(HEAA)を含む、請求項1〜13のいずれか一項に記載の印刷可能な組成物。
- 前記光重合開始剤が、2−ヒドロキシ−4’−(2−ヒドロキシエトキシ)−2−メチルプロピオフェノン、エチル(2,4,6−トリメチルベンゾイル)フェニルホスフィネート、フェニルビス(2,4,6−トリメチルベンゾイル)ホスフィンオキシド、ジフェニル(2,4,6−トリメチルベンゾイル)ホスフィンオキシド、またはそれらのうちの2種以上の組合せからなる群から選択される、請求項1〜15のいずれか一項に記載の印刷可能な組成物。
- 前記ビヒクルが、水溶性アクリルモノマーをさらに含む、請求項1〜16のいずれか一項に記載の印刷可能な組成物。
- 前記水溶性アクリルモノマーが、カルボキシエチルアクリレート、ポリ(アルキレン)(メタ)アクリレート、HEAA、HEMA、HEA、ACMO、NVP、V−CAP、またはそれらのうちの2種以上の組合せからなる群から選択される、請求項17に記載の印刷可能な組成物。
- 前記ビヒクルが、カルボン酸をさらに含む、請求項1〜18のいずれか一項に記載の印刷可能な組成物。
- 前記カルボン酸が、酢酸、プロパン酸、ブタン酸、ペンタン酸、ラウリン酸、ステアリン酸、デオキシコール酸、グルタミン酸、グルクロン酸、またはそれらのうちの2種以上の混合物からなる群から選択される、請求項19に記載の印刷可能な組成物。
- 前記カルボン酸が、約5mMから約200mMまでの濃度で存在する、請求項20に記載の印刷可能な組成物。
- 前記ビヒクルが、少なくともpH2を有する、請求項1〜21のいずれか一項に記載の印刷可能な組成物。
- 9−アンチルメチルN、N−ジエチルカルバメート、(E)−1−ピペリジノ−3−(2−ヒドロキシフェニル)−2−プロペン−1−オン、1−(アントラキノン−2−イル)エチルイミダゾールカルボキシレート、2−ニトロフェニルメチル4−メタクリロイルオキシピペリジン−1−カルボキシレート、1,2−ジイソプロピル−3−[ビス(ジメチルアミノ)メチレン]グアニジニウム−2−(3−ベンゾイルフェニル)プロピオネート、1,2−ジシクロヘキシル−4,4,5,5−テトラメチルビグアニジウムN−ブチルトリフェニルボレート、(Z)−{[ビス(ジメチルアミノ)メチリデン]アミノ}−N−シクロヘキシル(シクロヘキシルアミノ)メタニミニウムテトラキス(3−フルオロフェニル)ボレート、またはそれらのうちの2種以上の組合せからなる群から選択される光塩基発生剤をさらに含む、請求項1〜22のいずれか一項に記載の印刷可能な組成物。
- 約300nm〜約450nmの間の波長で0.1超の吸光度単位を有する吸収剤をさらに含む、請求項1〜23のいずれか一項に記載の印刷可能な組成物。
- 前記吸収剤が、タートラジンである、請求項1〜24のいずれか一項に記載の印刷可能な組成物。
- 約1ミクロン未満の解像度で印刷可能である、請求項1〜25のいずれか一項に記載の印刷可能な組成物。
- 約0.1mm/10mJ/cm2から約5mm/100mJ/cm2までのフォトスピード(Dp/Ec)で印刷可能である、請求項1〜26のいずれか一項に記載の印刷可能な組成物。
- 乾燥後の前記グリーンストレングスが、約5kPaから約10kPaまでである、請求項1〜26のいずれか一項に記載の印刷可能な組成物。
- 三次元細胞受容足場を調製する方法であって、請求項1〜28のいずれか一項に記載の印刷可能な組成物をプリントして、前記三次元細胞受容足場を組み立てるステップを含む、方法。
- 前記プリントするステップが、器官の前記三次元細胞受容足場を組み立て、前記器官は、哺乳動物の器官である、請求項29に記載の方法。
- 前記プリントするステップが、インクジェットプリント、押出成形プリント、またはレイヤーバイレイヤープリントを含む、請求項29または30に記載の方法。
- 三次元細胞受容足場を製造する方法であって:
印刷可能な組成物の層を面に堆積させて堆積層を得るステップ;
前記堆積層を照射するステップ;および
前記堆積層が前記三次元細胞受容足場を形成するまで前記堆積ステップと照射ステップを繰り返すステップ、
を含み、
前記印刷可能な組成物が、前記印刷可能な組成物の約0.3重量パーセントから約3.0重量パーセントまでの量で存在するコラーゲン;モノマー;光重合開始剤;およびビヒクル、
を含む、方法。 - 前記印刷可能な組成物が、請求項1〜28に記載の印刷可能な組成物である、請求項32に記載の方法。
- 前記堆積層が、印刷時に解像度100ミクロン以下を有する、請求項32または33に記載の方法。
- 前記堆積層が、約240から約405nmまでの波長で照射される、請求項32〜34に記載の方法。
- 前記波長が、約270nmまたは355nmである、請求項35に記載の方法。
- 堆積させるステップの前に、前記印刷可能な組成物を0℃〜約30℃の温度に加熱するステップをさらに含む、請求項32〜36のいずれか一項に記載の方法。
- 前記三次元足場を、細胞、組織、増殖因子、ラミニン、エラスチン、フィブロネクチン、プロテオグリカン、ヒアルロン酸、多糖類、またはそれらのうちの2つ以上の混合物と接触させるステップをさらに含む、請求項32〜37のいずれか一項に記載の方法。
- 請求項32に記載の方法によって作製される、三次元細胞受容足場。
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