JP2019528795A - 細胞培養における化学的微小環境のインビトロでの制御のための組成物、装置及び方法 - Google Patents
細胞培養における化学的微小環境のインビトロでの制御のための組成物、装置及び方法 Download PDFInfo
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Abstract
Description
a)酸素の還元を触媒する傾向がある1以上の酵素を含む溶液を調製する;
b)架橋剤を添加する;
c)溶液をゲル化する。
a)酸素の還元を触媒することができる1つ以上の酵素を含む溶液を調製する;
b)架橋剤を添加する;
c)溶液をゲル化する。
グルコースオキシダーゼ‐GOx‐は、以下の反応を触媒するオキシドレダクターゼファミリーの酵素である。
我々の装置でゲルによって生成された酸素の濃度勾配を評価するために、我々は走査型電気化学顕微鏡、すなわちマイクロメートルスケールで化学プロセスを評価するための、次いで細胞培養の微小環境を特徴付けるのに有用である、強力な装置を使用した。超微小電極(UME)を使用することによって、レドックスプロセスなどの電気化学的プロセスを検出することが可能である。プローブ(UME)は、3つのエンジンと3つの圧電素子とを有するシステムに接続され、次いで3つの直交軸X、Y及びZに移動して位置決めすることができる。このようなシステムはUMEで測定されたプロセスを空間的に解くことを可能にし、次いでトポグラフィーを表示し、基板の局所的な化学反応性を調べることを可能にする。まず、アプローチにより、一つが基板に近づくと、負のフィードバックモード、本発明者らの分析の場合では電流は、近づくにつれて徐々に減少し、d→0のようにそれは→0となる。ここで、dはプローブと特定の場合にはプラスチック又はガラスで作られた基板との間の距離である。このようにして、その機能性を測定するためにゲルに非常に近いが、機械的損傷を回避するために多すぎることなく、我々の電極にとって理想的な位置を選択することが可能である。この時点で、基板に対して平行に走査することによって、基板生成‐先端収集(SG‐TC)モードで、電極を表面近くに移動させ、溶液に浸し、UMEに流れる電流を基板上の各位置について記録する。それが観察されるのは、UMEで測定しているスピーシーズの濃度変化を反映する電流の変化である。プローブ位置に応じて、電流の曲線と二次元画像の両方を得ることができる。この酸素勾配を評価することに成功するという事実は、模倣したい腫瘍モデル又は生理学的/病理学的組織又はニッチ(例えば、幹細胞ニッチ)のモデルと同様のインビトロモデルを作成するためにパラメータを確立することによって、酵素濃度を再現された勾配と相関させることができ、デバイス基板の特定の位置にある細胞がどの酸素濃度にさらされているかを評価できること(すなわち各単一細胞の微小環境が測定されること)が基本となる。
試薬表
酸素勾配を測定するために使用される機器は、プローブ走査型電気化学顕微鏡「CH Instrument Texas」モデルCHI B910である。
本発明者らのヒドロゲルが細胞増殖にどのように影響したかを評価するために、MCF7及びMCF10Aの両方を以下のように培養した:
i)酵素を含むヒドロゲルの存在下、
ii)酵素を含まないヒドロゲルの存在下、白色、
iii)ヒドロゲルが存在しない場合
細胞の生存率の比色マーカーを用いて24、48及び72時間に細胞計数を行った。i)とii)との間の比較は、細胞が酵素によって生成された勾配に罹患しているかどうかを評価するための基本であり、ii)とiii)との間の比較は、ゲルの塩基組成が細胞に対していくらか毒性であるかどうかを評価するために行われた。
−分割(皿10センチ)
−ファルコン15に培地1mlを入れる
−培地を除去し、5mlのPBSで洗浄する
−1mlのトリプシン*と5分間のインキュベーター内
−トリプシンで細胞を再開し、それらをファルコン15に入れる
−2mlの培地を用いて細胞を皿に残したままにする
−培地3mlを加えて遠心する
−上清を除去し、数mlの培地に「ペレット」を再懸濁する(利用可能な細胞数に応じて)
−細胞計数
−64室で1:2細胞:PBSに希釈する(この手順のために約20μLの細胞懸濁液を使用する)
−PBS:ERYTHROSINEBで1:2溶液を希釈
−Burkerチャンバー内のそのように調製した溶液に10μlを入れ、光学顕微鏡でカウントを進める。
M×V×10−4×F.D.PBS×F.D.EB
ここで、MはBurckerチャンバーの細胞の細胞数の平均、Vは細胞のペレットが再懸濁された体積、F.D.は希釈係数である。
−プレーティング
−適切な量の細胞溶液を拾い上げ、それを適切な培地1mlに懸濁する。
−マルチウェル又は使用されている3.5cm幅のペトリ皿のすべてのチャンバーに再分配する。
*トリプシンはペプチド結合の破裂を促進し、細胞のペトリ皿への接着を可能にする。
細胞MCF10A及びMCF7をその後プレートすることができる、いくつかのプレート(皿)を調製した。2日間の培養後、コンフルエントに達する前に細胞を固定し、全ての点でその相対的計数を行い、そしてゲルからの異なる距離での増殖を評価した。
図3:対照プレート及び活性のない(酵素を含まない)タンパク質マトリックスで修飾したプレート上の細胞MCF10A及びMCF7の増殖曲線。曲線は、使用されたマトリックスの生体適合性及び毒性の非存在を示す。
Claims (24)
- マトリックスを含むインビトロ細胞培養物中で使用するための組成物であって、前記マトリックスが前記細胞培養物中の酸素勾配が制御される酸素の還元を触媒することができる1種以上の酵素を含むことを特徴とする、上記組成物。
- 前記マトリックスがポリマー又はタンパク質マトリックスである、請求項1に記載の組成物。
- 前記酵素が、グルコースオキシダーゼ(GOx)、NADPHオキシダーゼ、キサンチンオキシダーゼ、乳酸オキシダーゼ、チトクロームオキシダーゼ又はラッカーゼから選択される、請求項1又は2に記載の組成物。
- カタラーゼ酵素をさらに含む、請求項1〜3のいずれか一項に記載の組成物。
- 前記酵素が、架橋剤(クロスリンカー)によって前記マトリックスに共有結合している、請求項1〜4のいずれか一項に記載の組成物。
- 前記架橋剤が、グルタルアルデヒド(GDA)、ビス(スルホスクシンイミジル)スベレート、N‐ヒドロキシスクシンイミド、ホルムアルデヒド、光反応剤から選択される、請求項5に記載の組成物。
- 前記酵素が、機械的及び/又は静電的相互作用を介してポリマーマトリックス中に捕捉されている、請求項1〜4のいずれか一項に記載の組成物。
- 前記組成物がヒドロゲルである、請求項1〜7のいずれか一項に記載の組成物。
- 前記ヒドロゲルが、シリコーンヒドロゲル、ポリアクリルアミド、セルロース、セルロース誘導体、コラーゲン、カルボキシメチルセルロース、アルギン酸塩、キトサン、寒天、ポリマコン、ヒアルロン酸、ポリメチルメタクリレート、ヒドロゲルペプチド模倣物から選択される、請求項1〜8のいずれか一項に記載の組成物。
- 前記ヒドロゲルのマトリックスが、ウシ血清アルブミン(BSA)をさらに含む、請求項8又は9に記載の組成物。
- 前記ヒドロゲルのポリマーマトリックスが、グルタルアルデヒドにより共有結合したグルコースオキシダーゼ(GOx)、カタラーゼ及びウシ血清アルブミン(BSA)を含む又はからなる、請求項10に記載の組成物。
- 細胞用の容器と請求項1〜11のいずれか一項に記載の組成物とを含む、インビトロ細胞を培養するための装置。
- 前記容器がペトリ皿である、請求項12に記載の装置。
- 前記組成物が前記容器の底部に堆積されて1つ又は複数の層を形成する、請求項12又は13に記載の装置。
- 細胞培養物の化学的微小環境を制御し、及び/又はインビボ細胞の生理学的又は病理学的条件を模倣するためのインビトロ方法であって、細胞用容器中で細胞株又は初代細胞を培養する工程を特徴とし、請求項1から11のいずれか一項の組成物が寄託されている、上記方法。
- 前記組成物によって生成された酸素勾配を測定するさらなるステップを含み、特に、前記測定が走査型電気化学顕微鏡によって行われる、請求項15に記載の方法。
- 前記細胞培養物が癌細胞である、請求項15又は16に記載の方法。
- 前記細胞が上皮細胞である、請求項15又は16に記載の方法。
- 請求項1から10のいずれか一項に記載の組成物が堆積されている細胞用容器内で前記細胞株を培養する工程、及び前記細胞株に前記薬物又は化合物を添加する次の工程を含む、生理学的又は病理学的状態に対する薬物又は化合物の効果を評価するインビトロ方法。
- 前記組成物のマトリックスがミクロスフェアに封入されている、請求項15から19のいずれか一項に記載の方法。
- 前記微小球が磁性である、請求項20に記載の方法。
- 以下の工程を含む、請求項8から11のいずれか一項に記載のヒドロゲルの形態の組成物の調製方法:
a)酸素の還元を触媒することができる1つ以上の酵素を含む溶液を調製する;
b)架橋剤を添加する;
c)溶液をゲル化する。 - 前記架橋剤がグルタルアルデヒド(GDA)である、請求項1〜15のいずれか一項に記載の方法。
- BSAが添加される追加の工程を含む、請求項22又は23に記載の方法。
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