JP2020516311A - 細胞培養および生物医学的応用のためのヒドロゲル - Google Patents
細胞培養および生物医学的応用のためのヒドロゲル Download PDFInfo
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Abstract
Description
本願は、2017年4月10日に出願された米国特許出願第62/483,831号の優先権を主張し、その内容はその全体が参照により完全に本明細書に組み込まれる。
米国特許出願第2008/0220526号は、細胞培養表面のためのコーティングに関する。より詳細には、本発明は、天然由来のゴム、植物ゴム、ガラクトマンナンゴムまたはそれらの誘導体を含む、ゴムに由来するかまたはゴムを含有する細胞培養表面のためのコーティングに関する。本発明はまた、そのようなコーティングを有する製造品(例えば、細胞培養容器および実験器具)、これらのコーティングを細胞培養表面に適用する方法、およびコーティングされた細胞培養容器を使用する方法に関する。
1%w/vジェランガム溶液を調製する。4:1比率(v/v)でジェランガム溶液を細胞懸濁液と混合する。混合直後にゲル化が開始し、G’の増加を示す。G’>50paを示すまで、15分間、ソフトゲルを形成させることができる。ベータTC3細胞は、ヒドロゲル内で懸濁される。その後、ソフトゲルの上部に追加のDMEM培地を加え、ヒドロゲルは、G’の増加を示すことによってさらに安定化される。ハードゲルは、2時間(または一晩)後に形成し、500Paよりも高いG’を示す。ベータTC3細胞は、ヒドロゲル中で増殖でき、3日後に3Dコロニーを形成する。
1%w/vジェランガム溶液を調製する。1:1比率(v/v)でジェランガム溶液を細胞懸濁液と混合する。混合直後にゲル化が開始し、G’の増加を示す。G’>50paを示すまで、15分間、ソフトゲルを形成させることができる。Ins−1細胞は、ヒドロゲル内で懸濁される。その後、ソフトゲルの上部に追加のRPMI培地を加え、ヒドロゲルは、G’の増加を示すことによってさらに安定化される。ハードゲルは、2時間(または一晩)後に形成し、300Paよりも高いG’を示す。Ins−1細胞は、ヒドロゲル中で増殖でき、3日後に3Dコロニーを形成する。
Claims (40)
- 1種以上の水溶性高アシルジェランガムポリマーと、
1種以上の水溶性低アシルジェランガムポリマーと、
1種以上の水溶性化学修飾ジェランガムポリマーまたは1種以上のペプチド修飾ジェランガムポリマーと、を含む、多糖ヒドロゲルのための組成物。 - 形成された前記多糖ヒドロゲルが注入用途に適したソフトゲルである、請求項1に記載の組成物。
- 前記多糖類ヒドロゲルをせん断力によって液体状態に変換させるか、または前記せん断力が一旦停止するとそのヒドロゲル状態を回復させることによって、前記ソフトゲルが、せん断減粘および自己修復レオロジー特性を示す、請求項2に記載の組成物。
- 前記せん断力が、ピペット操作、シリンジ注入、もしくはポンプ灌流、またはこれらの組み合わせによって働く、請求項3に記載の組成物。
- 形成された前記多糖ヒドロゲルがハードゲルである、請求項1に記載の組成物。
- 前記ハードゲルがピペット操作またはせん断により破壊されると、前記ハードゲルがより小さなゲル粒子に分解し、そして1種以上の生物活性分子についての親和性を有するようなレオロジー特性を有する3−Dゲル構造を前記ハードゲルが示す、請求項5に記載の組成物。
- 前記ハードゲルが、約10Paよりも大きい貯蔵弾性率値を有する、請求項5に記載の組成物。
- 前記ハードゲルが約80℃以下の温度でそのゲル形成を維持するが、外力で乱された場合にはより小さなゲル粒子に分解することが可能である、請求項5に記載の組成物。
- 前記1種以上の化学修飾ジェランガムポリマーが、
a)天然または合成由来のポリマー、化学修飾ポリマーまたはコポリマー、ヒアルロン酸塩、キトサン、コラーゲン、ポリエチレングリコール抗凝固剤、造影剤、化学療法剤、およびシグナル伝達経路分子からなる群より選択される有機分子、ならびに
b)生物活性ガラス、ヒドロキシアパタイト、リン酸カルシウムおよび鉄からなる群より選択される無機分子、からなる群より選択される、請求項1に記載の組成物。 - 前記1種以上の生物活性分子が、加熱前にジェランガム溶液に加えられ、細胞、ペプチド、タンパク質、脂質、多糖類、成長因子、成長ホルモン、抗体、酵素、細胞受容体、細胞リガンド、抗生物質、抗微生物剤、抗菌剤、抗真菌剤、RGD、IKVAV、REDV、YIGSRY、ポリリジンを含む、NH2基、COOHおよびCONH2基を有する機能性ペプチド分子からなる群より選択される、請求項6に記載の組成物。
- 前記ソフトゲルは、余分なリン酸緩衝液、細胞培養培地、もしくはイオン溶液、またはそれらの組み合わせの水溶液に前記ソフトゲルを浸すことにより、ハードゲルに変換される、請求項2に記載の組成物。
- 1種以上の生物活性分子が、それらの生物活性を維持しながら、前記多糖ヒドロゲル系に接触、付着、懸濁、埋め込み、または捕捉されている、請求項1に記載の組成物。
- 前記1種以上の生物活性分子が、それらの生物活性を維持しながら、前記多糖ヒドロゲルに懸濁または捕捉されている、請求項12に記載の組成物。
- 前記1種以上の生物活性分子が、前記1種以上の生物活性分子の生物活性を維持しながら、前記多糖ヒドロゲルに接触、付着、懸濁、埋め込み、または捕捉されている、請求項5に記載の組成物。
- 前記1種以上の生物活性分子が、前記1種以上の生物活性分子の生物活性を維持しながら、前記多糖ヒドロゲルに懸濁または捕捉されている、請求項14に記載の組成物。
- ペプチドによって修飾されたジェランガムの溶液は、混合物としての前記ジェランガム溶液にペプチドを加え、次いで約100℃以上の温度および約1psi〜約40psiの圧力で、約3〜約30分の時間の間加熱することによって形成される、請求項1に記載の組成物。
- 前記組成物は、約0.001%〜約20%の前記1種以上の高アシルジェランガムポリマー、約0.001%〜約20%の前記1種以上の低アシルジェランガムポリマー、約0.001%〜約20%の前記1種以上の修飾ジェランガムポリマーを含み、約0.00001%〜約30%の前記1種以上の生物活性分子をさらに含む、請求項1に記載の組成物。
- 前記組成物が約10Paの貯蔵弾性率値を有する、請求項17に記載の組成物。
- 前記組成物は、約0.01%〜約10%の前記1種以上の高アシルジェランガムポリマー、約0.01%〜約10%の前記1種以上の低アシルジェランガムポリマー、約0.01%〜約10%の前記1種以上の修飾ジェランガムポリマーを含み、約0.001%〜約20%の前記1種以上の生物活性分子をさらに含む、請求項1に記載の組成物。
- 前記組成物が約10〜約20000Paの貯蔵弾性率値を有する、請求項20に記載の組成物。
- 請求項1に記載の多糖ヒドロゲルを形成するための方法であって、
水溶性ジェランガムポリマーを、約4℃〜約99℃の範囲の温度で0.001%w/vより高い固形分を含む水ベースの溶媒に溶解する工程と、
前記溶液を約100℃以上の温度および約1psi以上の圧力で3分間以上加熱する工程と、
溶液を、リン酸緩衝液(PBS)、細胞培養培地またはイオン溶液と直接混合して前記多糖ヒドロゲルの形成をトリガーすることによって、約4℃〜約60℃の範囲の温度で網状化させる工程であって、
前記多糖ヒドロゲルの貯蔵弾性率(G’)は混合の際に増加し、30分間以内に約10Paを超え、その結果、前記系が3D増殖のためにそのヒドロゲルマトリックス内に懸濁した生物活性分子を維持する、網状化させる工程と、
化学物質または生物活性分子が、形成された前記多糖ヒドロゲルに接触、付着、懸濁、埋め込み、または捕捉されるように、前記化学物質または生物活性分子を加える工程と、を含む、方法。 - 前記水ベースの溶媒が、水、リン酸緩衝溶液(PBS)、生理食塩水、および細胞培養培地を含む、請求項21に記載の方法。
- 前記固形分が約0.001%(w/v)〜10%(w/v)の範囲の量で使用される、請求項21に記載の方法。
- 前記溶液と約0.01%(w/v)以上のイオン濃度のトリガー溶液とが約100:1〜約1:1の比率範囲で混合されると、ソフトヒドロゲルが形成される、請求項21に記載の方法。
- 前記溶液と約0.01%(w/v)の高イオン濃度のトリガー溶液とが約1:1〜約1:100の比率範囲で混合されると、ハードヒドロゲルが形成される、請求項21に記載の方法。
- 前記溶液と約0.01%(w/v)の通常のイオン濃度のトリガー溶液とが約1:1〜約1:20の比率範囲で混合されると、ハードヒドロゲルが形成される、請求項21に記載の方法。
- 前記溶液が、網状化の前に、約100℃〜約132℃、好ましくは約100℃〜約121℃の範囲の温度に加熱される、請求項21に記載の方法。
- 前記溶液が、網状化の前に、約1psi〜約25psiの範囲の圧力下で加熱される、請求項21に記載の方法。
- 前記溶液が、網状化の前に、約1psi〜約15psiの範囲の圧力下で加熱される、請求項21に記載の方法。
- 前記溶液が、網状化の前に、約1分〜約30分間加熱される、請求項21に記載の方法。
- 前記溶液が、網状化の前に、約5分〜約20分間加熱される、請求項21に記載の方法。
- 前記形成されたソフトゲルが、細胞培養培地の水溶液またはイオン溶液に浸されることにより、ハードゲル系に変換される、請求項11に記載の組成物。
- 請求項11に記載の方法により形成され相互変換されたヒドロゲル系であって、前記生物活性分子は、前記ジェランガム溶液またはトリガー溶液と混合することにより、ヒドロゲル形成の前もしくは後、またはヒドロゲルと周囲の培地との交換の前もしくは後に前記ヒドロゲル系に加えることができる、ヒドロゲル系。
- 請求項1に記載の組成物に由来する、多糖ヒドロゲルの使用であって、細胞生存率アッセイ、生死アッセイ、ハイスループットスクリーニング、蛍光染色および画像化、組織学的分析、ならびに3Dバイオプリンティングを含む、創薬および生物医学的応用向けの汎用プラットフォームとしての、使用。
- 生細胞が、前記ヒドロゲルの上部で増殖するか、または前記ヒドロゲルに埋め込まれ、カプセル化され、そして前記ヒドロゲルを破壊すること、およびDI水または低イオン濃度溶液で前記ヒドロゲルを溶解することによって、ヒドロゲル系から回収される、請求項34に記載の使用。
- 前記ヒドロゲル中でのインビトロ3D細胞培養のための2段階手順が提供され、前記手順は、
1)生細胞が、前記ヒドロゲルの形成前にジェランガム溶液または細胞培養培地などのトリガー溶液と混合され、前記細胞は前記ソフトヒドロゲル内で均質に懸濁され、ピペット操作または注入により個々の細胞培養プレートまたは様々な容器に移す準備ができていることと、
2)一旦、前記ソフトゲルを最終容器に配置したら、追加のトリガー溶液を前記ソフトゲルの上部に加えるか、または前記ソフトゲルを囲んで、前記ソフトゲルを前記ハードヒドロゲルに変換し、前記3Dマトリックス構造がさらに安定化され、栄養または他の生体分子が前記ヒドロゲル系に加えられ、前記ヒドロゲルと周囲の培地との間で交換されることと、を含む、請求項34に記載の使用。 - 2Dヒドロゲルコーティング細胞培養研究のための請求項34に記載の使用であって、前記ソフトヒドロゲルが前記培養プレートの表面に直接加えられて、前記細胞培養プレートをコーティングし、そして、生細胞をヒドロゲルの上部に加えて、2Dで増殖させることができ、一部の細胞は、上部からヒドロゲルに浸透し、3D構造として増殖できる、使用。
- 前記水溶性ジェランガムポリマーにクエン酸ナトリウムを加えて、ジェランガムポリマー溶液を形成する工程と、
前記ジェランガムポリマー溶液のpHを中性pHに調整する工程と、をさらに含む、請求項21に記載の方法。 - 前記ソフトヒドロゲルが、注入に適した繊維構造を含む、請求項24に記載の方法。
- 前記ハードヒドロゲルが凝集構造を含む、請求項25に記載の方法。
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WO2022040959A1 (zh) * | 2020-08-26 | 2022-03-03 | 基可生医股份有限公司 | 3d细胞培养胶体的套组及其用于3d细胞培养的方法 |
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