1。アッセイセットアップ<ol><li> 4℃（約2時間）で氷の上でマトリゲルの小アリコート（&lt;500μl）を解凍します。 </li><li>ゲルのレシピ（下の表に例のボリュームを参照してください）​​準備：10倍PBS（総量で1×）、1N水酸化ナトリウム（追加されたコラーゲンの0.023ボリュームと同等、またはコラーゲンの製造業者の推奨に従って、必要に応じて）、マトリゲルとコラーゲンにIを入力します。に1 mg / mlとそれぞれ1.3 mg / mlの最終濃度（他のマトリックス製剤は細胞の種類や実験に応じて使用されるかもしれません）。 </li></ol> 例のゲルのレシピ <table border="1"><tbody><tr><td> コンポーネント </td><td> ストック濃度 </td><td> 最終濃度 </td><td> ボリュームを加える </td></tr><tr><td> 10×PBS </td><td> 10X </td><td> 1X </td><td> 0.090ミリリットル</td></tr><tr><td>滅菌水</td><td></td><td></td><td> 0.346ミリリットル</td></tr><tr><td> 1NのNaOH </td><td></td><td></td><td> 0.008ミリリットル</td></tr><tr><td>マトリゲル</td><td> 9.90 mg / mlの</td><td> 1 mg / mlの</td><td> 0.101ミリリットル</td></tr><tr><td>ラット尾I型コラーゲン</td><td> 3.66 mg / mlの</td><td> 1.3 mg / mlの</td><td> 0.355ミリリットル</td></tr></tbody></table><ol start="3"><li> 4℃で1時間上記のようにインキュベートし、最終的なソリューションと同じ順序で氷の上でゲルのコンポーネントを混在させる我々の経験では、コラーゲンのより均一なゲル化における細胞播種の結果の前に1時間インキュベーション。すべての回で氷の上でマトリゲルやコラーゲンを維持するとゲル化を防ぐためにできるだけ速く動作することを確認してください。 </li><li>滅菌ピンセットを用いて12ウェルプレートに配置する12ミリメートルの直径8μmの孔の細胞培養挿入します。 </li><li> 5×10 6細胞/ ml（総ボリュームはゲル溶液の10％でなければなりません）で無血清培地で細胞を再懸濁しをカウントします。 </li><li>細胞のSUの100μlを加えゲル溶液900μL（最終細胞濃度5×10 5細胞/ ml）にspensionと上下に穏やかにピペッティングにより混和します。 </li><li>ゲルが重合されるまで30分間、37℃、5％CO 2インキュベーターにそれぞれ挿入し、転送に最終混合物を150μlのを追加します。 </li><li>無血清培地を使用して、 </li></ol><ul><li>ゲルの上に100μlと静止状態のインサート下の1200を添加する。井の中の挿入および外側内の流体のレベルは、ゲル間の最小限の圧力差としない間流れの中で、その結果、ほぼ等しくなければなりません。 </li><li> FLOW条件のゲル上での挿入、650μlの下に100μlを添加します。彼らはこれらの場所では膜を通って移行するから細胞を防ぐことができますように、インサートの下に気泡を避けるように注意してください。これらのボリュームによって生成された圧力差は約1.3 CM H 2 O（または1ミリメートルHg）である。 </li></ul><ol start="9"><li> 37にプレートを置きます°C、24時間、5％CO 2インキュベーター。 </li></ol> 2。細胞は、染色と計数<ol><li> 500の1X PBSを添加ウェルあたりに新しい24ウェルプレートを追加し、これはインサートを洗うために使用されます。 </li><li>フローのトランスウェルの上部に残っている培地を除去し、ボリュームを決定します。もともとは、650μlを（これは下記を参照してください、流量の計算に使用されます）追加された合計ボリュームから、残りのボリュームを差し引くことにより、総溶出体積を計算します。インサートからゲルを削除するには、非浸潤細胞を除去するために膜の最表面を拭くために綿棒を使用しています。インサートを洗うために15秒間の1X PBSを含む24ウェルプレートへの挿入を配置します。 </li><li> PBSを除去し、各インサートの下に4％パラホルムアルデヒド（PFA）500μlを追加して、転生細胞を固定し、室温で30分間インキュベートします。 </li><li> PFAを削除し、残留固定を削除するには、500μlの1X PBSで一回すすいでください。 </li><li> 500μlのoを追加するfを0.5％トリトンX-100に挿入下に解決し、細胞を透過処理するために室温で10分間インキュベートします。 </li><li> （転生細胞が下向き）膜面の下側を下に置くように注意して、1X PBS溶液中で2μg/ mlのDAPIの100μl中にカミソリの刃と場所を使用して、インサートの膜を切り取ります。 </li><li>室温で30分間150rpmでシェーカー上にアルミ箔と場所のプレートをラップします。 </li><li>無料のDAPIを削除するには、シェーカー（リピート10分ごとに3回）に500μlの1X PBSで膜を洗浄します。 </li><li>上向きに転生細胞を用いたスライドガラスの上に置き膜は、マウントソリューションとカバースリップを追加します。 </li></ol> 3。データ解析<ol><li>各膜の5ランダムに選択された場所（端から離れて滞在し、10倍または20倍の対物レンズを使用）にDAPIで染色した核をカウントします。 </li><li>平均細胞数を計算し、以下の式を使用して、パーセントの浸潤を取得します。 パーセント侵略= 100％×（平均細胞数xの膜面積）/（細胞播種の顕微鏡画像のx番号の表面積） パーセントの浸潤は独立した実験間の比較を可能にするためにいくつかの制御条件（通常は静的な状態）に正規化することができます。 </li><li>平均流速を計算します。インキュベーション時間（例えば24時間）することによって、フロー状態の総溶出ボリュームを分割します。 </li><li>平均流速を計算します。ゲル/メンブレン（このケースでは60ミリメートル2）の断面積で平均流量を分割します。 </li></ol> 4。代表的な結果間流の下で腫瘍細胞の浸潤を測定するために、我々は、MDA-MB-435S転移性悪性黒色腫細胞を用いた我々の3次元流れの浸潤アッセイを行った。これらの細胞は、以前に間質液の流れ13から14に対応して侵入することが示されている。細胞は、1.3 mg / mlのラット太極拳からなるマトリックスに埋め込まれていたlの腱のコラーゲンI型および5×10 5細胞/ mlの最終細胞濃度が1 mg / mlのマトリゲル基底膜マトリックス。二つの異なる条件を比較した：（1）平均的な間隙流量= 0.1μmのs -1の（2）静的状態= noを測定可能な流量（ 図1）。 24時間後、膜の孔を通って侵入した細胞は、細胞計数を容易にするためにDAPIで染色した。 図2は、浸潤細胞の代表的なイメージを示しています。視野の下で、膜の孔のみが表示されています。蛍光を使用して、DAPI染色した核が細胞計数のために使用されたとファロイジン染色されたF-アクチンの構造は、細胞体（オプション）を可視化するために使用された。等分散を仮定し、スチューデントのt検定を用いて、我々はその間隙流れが大幅に静的な条件で2.3倍でMDA-MB-435S細胞浸潤（P = 0.003）（ 図3）が増加した。このcorroboraこの細胞株13から14を使用して同様の所見を（ただし、別の行列の条件、したがって、流速を含む）tesの。 <img alt="図1" src="/files/ftp_upload/4159/4159fig1.jpg" /> 図1 3-D間質液の流れ浸潤アッセイの模式図。最初に適切な濃度とボリュームを使用してゲル溶液を準備します。その後ゲル溶液にセルを追加し、細胞培養インサートに転送されます。最後に、各条件にメディアの適切な量を追加し、インキュベートします。間質液の流れが流体圧力ヘッドによって駆動されます。 <img alt="図2" src="/files/ftp_upload/4159/4159fig2.jpg" /> 図2膜上のMDA-MB-435S細胞を転生。細胞が浸潤細胞のカウントを容易にするためにDAPIおよびAlexa Fluor 488標識ファロイジンで私たちの間質液の流れ浸潤アッセイで染色した後に修正された侵略、明視野下の膜の）写真は、B）DAPI STAINED核（青）、C）のAlexa Fluor®488ファロイジン（緑色）F-アクチン染色した。スケールバーは50μmを表す。 <img alt="図3" src="/files/ftp_upload/4159/4159fig3.jpg" /> 図3は、フローの下でMDA-MB-435S細胞の浸潤を増加しました。 24時間後の細胞の浸潤が有意に間流れた（p = 0.003）によって強化されています。結果は、平均的な静的な状態に正規化され、値は6セルカルチャーインサートの平均±SEMを表しています。

<ol>
	<li>Cichon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+influences+that+drive+progression+from+benign+breast+disease+to+invasive+breast+cancer.">Microenvironmental influences that drive progression from benign breast disease to invasive breast cancer.</a> J. Mammary Gland. Biol. Neoplasia. 15, 389-3897 (2010).</li><li>Proia, D. A., Kuperwasser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroma:+tumor+agonist+or+antagonist.">Stroma: tumor agonist or antagonist.</a> Cell Cycle. 4, 1022-1025 (2005).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment+and+progression.">Tumor microenvironment and progression.</a> J .Surg. Oncol. 103, 468-474 (2011).</li><li>Provenzano, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC Med. 6, 11 (2008).</li><li>Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Butler, T. P., Grantham, F. H., Gullino, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+transfer+of+fluid+in+the+interstitial+compartment+of+mammary+tumors.">Bulk transfer of fluid in the interstitial compartment of mammary tumors.</a> Cancer Res. 35, 3084-3088 (1975).</li><li>Dafni, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+vascular+endothelial+growth+factor+165+drives+peritumor+interstitial+convection+and+induces+lymphatic+drain:+magnetic+resonance+imaging,+confocal+microscopy,+and+histological+tracking+of+triple-labeled+albumin.">Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: magnetic resonance imaging, confocal microscopy, and histological tracking of triple-labeled albumin.</a> Cancer Res. 62, 6731-6739 (2002).</li><li>Chary, S. R., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+interstitial+convection+and+diffusion+of+albumin+in+normal+and+neoplastic+tissues+by+fluorescence+photobleaching.">Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 5385-5389 (1989).</li><li>Heldin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+interstitial+fluid+pressure+-+an+obstacle+in+cancer+therapy.">High interstitial fluid pressure - an obstacle in cancer therapy.</a> Nat. Rev. Cancer. 4, 806-813 (2004).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li><li>Shields, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chemotaxis+as+a+mechanism+of+tumor+cell+homing+to+lymphatics+via+interstitial+flow+and+autocrine+CCR7+signaling.">Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling.</a> Cancer Cell. 11, 526-538 (2007).</li><li>Shieh, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+invasion+is+promoted+by+interstitial+flow-induced+matrix+priming+by+stromal+fibroblasts.">Tumor cell invasion is promoted by interstitial flow-induced matrix priming by stromal fibroblasts.</a> Cancer Res. 71, 790-800 (2011).</li><li>Shi, Z. D., Wang, H., Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+proteoglycans+mediate+interstitial+flow+mechanotransduction+regulating+MMP-13+expression+and+cell+motility+via+FAK-ERK+in+3D+collagen.">Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.</a> PLoS One. 6, e15956 (2011).</li><li>Fleury, M. E., Boardman, K. C., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+morphogen+gradients+by+subtle+interstitial+flow+and+matrix+interactions.">Autologous morphogen gradients by subtle interstitial flow and matrix interactions.</a> Biophys J. 91, 113-121 (2006).</li><li>Miteva, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmural+flow+modulates+cell+and+fluid+transport+functions+of+lymphatic+endothelium.">Transmural flow modulates cell and fluid transport functions of lymphatic endothelium.</a> Circ. Res. 106, 920-931 (2010).</li><li>Ng, C. P., Hinz, B., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+fluid+flow+induces+myofibroblast+differentiation+and+collagen+alignment+in+vitro.">Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro.</a> J. Cell. Sci. 118, 4731-4739 (2005).</li><li>Kunder, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+particles+deliver+peripheral+signals+to+remote+lymph+nodes.">Mast cell-derived particles deliver peripheral signals to remote lymph nodes.</a> J. Exp. Med. 206, 2455-2467 (2009).</li><li>Helm, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+interstitial+flow+and+VEGF+directs+capillary+morphogenesis+in+vitro+through+a+gradient+amplification+mechanism.">Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 15779-15784 (2005).</li><li>McGuire, P. G., Seeds, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+plasminogen+activator+with+a+reconstituted+basement+membrane+matrix+and+extracellular+macromolecules+produced+by+cultured+epithelial+cells.">The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells.</a> J Cell Biochem. 40, 215-227 (1989).</li><li>Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+type+IV+procollagen,+laminin,+and+heparan+sulfate+proteoglycan+from+the+EHS+sarcoma.">Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma.</a> Biochemistry. 21, 6188-6193 (1982).</li><li>Haessler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+dynamics+of+breast+cancer+cells+in+a+tunable+3D+interstitial+flow+chamber.">Migration dynamics of breast cancer cells in a tunable 3D interstitial flow chamber.</a> Integr. Biol. (Camb). , (2011).</li><li>Polacheck, W. J., Charest, J. L., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+flow+influences+direction+of+tumor+cell+migration+through+competing+mechanisms.">Interstitial flow influences direction of tumor cell migration through competing mechanisms.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 11115-11120 (2011).</li></ol>

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ここでは、細胞培養インサート内の3-Dマトリックス中に埋め込まれた細胞を用いて、腫瘍細胞の浸潤の間の流れの影響を定量化する方法を説明してきました。これと同様の方法は、細胞の種類13-15、17のさまざまな格子フローの効果を調べるために使用されている。我々のアプローチは、部分的にI型コラーゲンおよび上皮組織と周囲の間質21から22の基底膜に見られる蛋白質を...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>コラーゲン（ラットテール） </td><td> BD </td><td> 354236 </td><td>無菌に保つ</td></tr><tr><td> Millicellセルカルチャーインサート</td><td>ミリポア</td><td> PI8P01250 </td><td> 8μmの孔径、ポリカーボネート膜</td></tr><tr><td>マトリゲル</td><td> BD </td><td> 354234 </td><td>無菌に保つ</td></tr><tr><td> PBS </td><td>シグマアルドリッチ</td><td> 100M-8202 </td><td>洗浄ステップのためにゲル溶液、1xを調製するための10倍</td></tr><tr><td>水酸化ナトリウム、1.0Nソリューション</td><td>シグマアルドリッチ</td><td> S2770 </td><td>無菌に保つ</td></tr><tr><td> DMEM 1X </td><td> CellGro </td><td> 10から013-CV </td><td>無菌に保つ</td></tr><tr><td>ウシ胎児血清</td><td>アトランタバイオ</td><td> 511150 </td><td>無菌に保つ</td></tr><tr><td>ペニシリンストレプトマイシン</td><td> CellGro </td><td> 30002CI </td><td>無菌に保つ</td></tr><tr><td>トリトンX-100 </td><td>シグマアルドリッチ</td><td> X100-500ミリリットル</td><td> PBS中の0.5％ </td></tr><tr><td>パラホルムアルデヒド</td><td>フィッシャー·サイエンティフィック</td><td> 04042-500 </td><td> PBS中4％ </td></tr><tr><td>脱イオン水</td><td></td><td></td><td>無菌に保つ</td></tr><tr><td> 4 &#39;,6-diaminido -2 - フェニルインドール（DAPI） </td><td> MPのバイオメディカル</td><td> 0215757401 </td><td> 1 mg / mlのストック溶液</td></tr><tr><td>マウントソリューション</td><td>サーモフィッシャーサイエンティフィック</td><td> TA-030-FM </td><td></td></tr><tr><td>トリプシン-EDTA </td><td> CellGro </td><td> 25から052-CI </td><td>無菌に保つ</td></tr></tbody> &lt;/ TABLE&gt;

three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion

ほとんどの固形腫瘍の増殖と進行は、癌細胞と間質に関連した腫瘍微小環境1でシグナルへの応答の最初の変換に依存しています。以前に、腫瘍の微小環境の研究は主に腫瘍間質相互作用の1から2に焦点を当てている。しかし、腫瘍の微小環境にも影響をよくわかっていないまま生物物理学的勢力が多数含まれています。これらの力は、遺伝子発現の変化、細胞分裂、分化、浸潤3につながる腫瘍増殖の生体力学的な結果である。行列の密度4、剛性5-6、および構造6-7、間質液圧8、間質液の流れ8はすべての癌の進行中に変更されています。 特に間質液の流れが正常組織8-10に比べ腫瘍で高くなっています。推定間質液のFLフロー速度を測定し、腫瘍の大きさと分化9、11に応じて、0.1から3ミクロンs -1の範囲内であることがわかった。これは、腫瘍血管新生と血管透過性の増大12に起因する間質液圧の上昇によるものです。間質液の流れが癌細胞の浸潤13-14、血管の線維芽細胞と平滑筋細胞15を増加させることが示されている。この侵略は3-D 16でセルの周囲に作成またはマトリックスメタロプロテアーゼ（MMP）の発現15、ケモカインの分泌と細胞接着分子の発現17日増加した自己走勾配に起因することがあります。しかし、細胞は流体の流れを感知するメカニズムはよく理解されていません。腫瘍細胞の挙動を変更することに加え、間質液の流れが腫瘍微小環境内の他の細胞の活性を調節する。それは（）腫瘍促進myofibrに線維芽細胞の分化を駆動するに関連付けられていますoblasts 18、（b）の抗原とリンパ節19、及び（c）変調リンパ内皮細胞の形態20に他の可溶性因子の輸送。 ここで紹介する手法は、in vitroでの細胞の間質液の流れを課し、浸潤（ 図1）に及ぼす影響を定量化します。このメソッドは、間質と癌細胞の浸潤13-15、17日に流体の流れの効果を測定するために、複数の研究では公開されています。マトリックス組成物、細胞型、細胞濃度を変えることによって、このメソッドは、他の病気や浸潤、分化、増殖、遺伝子発現などの細胞プロセスの間の流れの影響を研究する生理学的システムに適用することができます。

Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

間質液の流れが固形腫瘍で上昇し、腫瘍細胞の浸潤を調節することができる。ここでは、マトリックス中に埋め込まれた細胞に間質液の流れを適用し、細胞浸潤に及ぼす影響を測定する手法を説明します。このテクニックは簡単に他のシステムを研究するために適応させることができます。

腫瘍細胞の浸潤を間質液流れに及ぼす影響を測定するための三次元細胞培養モデル

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

three-dimensional-cell-culture-model-for-measuring-effects

Research

JoVE Journal

Bioengineering

16.4K ビュー.  Drexel University. 間質液の流れが固形腫瘍で上昇し、腫瘍細胞の浸潤を調節することができる。ここでは、マトリックス中に埋め込まれた細胞に間質液の流れを適用し、細胞浸潤に及ぼす影響を測定する手法を説明します。このテクニックは簡単に他のシステムを研究するために適応させることができます。

ビデオ: Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巣と卵管の三次元器官培養用アルギン酸ゲル

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

生物工学

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

モデルの声帯ポリープによる三次元流分離の調査

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

癌細胞浸潤への応用による腫瘍間質微小環境の組織工学

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

三次元コラーゲンマトリックス内の細胞移動の解析

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

遺伝的に改変された細胞移植のためのマイクロパターン培養プレート上スフェロイド細胞に対する遺伝子導入

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

肝組織工学のための三次元細胞培養プラットフォーム：逆コロイド結晶ポリ（エチレングリコール）足場の作製

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

三次元培養細胞遊走を特徴付ける傷の環境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

ケラチン生成細胞増殖の 2 次元と 3 次元の評価タイプ I コラーゲン基板

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

均質な細胞分布のための三次元バイオプリンティングにおける多層ヒドロゲルバイオインクの使用

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

音響セパレータを用いたCHO採取細胞培養液の一次解明

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...