Name: utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells
Uploaded: 2014-12-07T14:00:00.000+00:00
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前立腺細胞株は、親切にがんセンター、カリフォルニア大学デ​​ービス校で博士陵王瑜博士興-寺院カンフーによって提供されます。このプロジェクトは、NIH電気走助成4R33AI080604によってサポートされています。

1.培養前立腺細胞<ol><li>培養PRNS-1-1、5％CO 2、37℃で、10％FBSおよび抗生物質-抗真菌を補充したRPMI 1640培地中で100mm培養皿上とPNT2前立腺細胞。細胞が電気走実験のために80％の集密度に達するまで、毎日の培地をリフレッシュします。 </li></ol> 2.組立電気走チェンバース<ol><li>下部チャンバーの組み立て<ol><li> 2-プロパノールでプラスチックの電気走室を拭きます。注射器でチャンバーの底側に円形の開口部周りのマリングレードのシリコンシーラーを適用し、大規模なカバーガラス（ 図1）を取り付け。カバーガラスを押し下げると綿棒で余分な接着剤をふき取るために綿棒の裏側を使用してください。円形の開口部は、媒体と、電流が2インナーメディア貯水池の間に位置し、細胞の上に流れるようになります。 </li><li> 6ミリメートル×2を作るために小さなカバーガラスをカットダイヤモンドポイントマーカーと5ミリメートルスペーサー。 </li><li>チャンバーを裏返し。注射器および/または下部ガラス上の2の円形開口部の間の細胞接着10ミリメートル×25mmのチャネル表面を作成するための金属スパチュラでシリコン接着剤の2ストライプを適用する。 2円形の開口部との間にガラススペーサの2枚を接着。スペーサーを押し下げると綿棒および/または爪楊枝で余分な接着剤をふき取るために綿のアプリケーターの裏側を使用してください。 </li><li>シリコン接着剤が硬化するまで、24時間チャンバーを乾燥させる。接着剤から酢酸残基を除去するために蒸留水で一晩チャンバーを浸す。すぐに使用するためのチャンバを乾かしたり、清潔​​な容器に後で使用するためにチャンバーを保存する。 <img alt="図1" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig1highres.jpg" width="500" /> 図1：底の電気走室の組み立て。 A）大カバーガラスを清浄室の底部に取り付けられている。B）Tガラススペーサの破片woは細胞が付着するための10ミリメートル×25 mmのチャンネルを作成するために2円形の開口部との間で接着されている。 </li></ol></li><li>電気走チャンバーに細胞を播種<ol><li>実験に必要な電気走室の必要とする数を準備します。各細胞株または処置は、単一電気走室を必要とする。 2-プロパノールとの電気走室を拭きます。滅菌PBSで3回チャンバーを洗浄し、チャンバ内の2円形の開口部との間の液体の流れを確認してください。 </li><li>無菌の細胞培養皿でチャンバを囲み、それらを15分間37℃で平衡化させる。 5分間37℃で5ミリリットル0.25％トリプシン-EDTAを使用して培養皿から前立腺細胞を剥離。 PBS中5ミリリットル、10％FBSをトリプシン-EDTAを中和する。 </li><li> 15ミリリットルチューブに細胞を移し、200×gで、37℃で5分間細胞を遠心する。上清を吸引し、培養培地1ml中に細胞を再懸濁する。 </li><li> 20μを取る計数チャンバーにロードし、細胞数を計算するために細胞溶液のリットル。培養培地で8×10 4細胞/ mlに細胞濃度を調整します。 </li><li> 37℃のインキュベーターから電気走室を取る。各チャンバーに細胞懸濁液の350μlのをシード。 </li><li>湿ったキムワイプまたは5％CO 2、37℃のインキュベーター内の湿度チャンバ内の培養皿に一晩チャンバー中で細胞をインキュベートする。 </li></ol></li></ol><img alt="図2" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig2highres.jpg" width="500" /> 図2：チャンバーに細胞を播種下部チャンバはA）前立腺細胞を、トリプシン処理し、計数し、チャンバーに移し、一晩インキュベートし、乾燥し、そして洗浄されるB）トップカバーガラスを撮影する前に、チャンバをシールするために装着される。 <ol start="3"><li> A上部チャンバーをssembling <ol><li>撮影の前に上部チャンバーを組み立てます。顕微鏡は、一度に単一の電気走チャンバを撮像収容することができる。各電気走室37℃で培養培地10mlを温める。 37℃の加温プレートにインキュベーターから電気走室を転送します。 </li><li>未付着細胞を除去し、培養培地で細胞を洗浄します。チャンバ内の培地を400μlのままにしておきます。両方のガラススペーサの上に高真空グリースを適用するために注射器を使用してください。 </li><li>チャンバーをシールするために小さなカバーガラスを追加します。静かに綿棒の裏側にカバーガラスを下に押してください。綿棒と過剰メディアを拭き取ってください。 </li><li>ガラス表面を乾燥させ、カバーグラスとチャンバとの間の隙間をシールするために高真空グリースを塗布。グリースを広めるために金属スパチュラを使用してください。インナー貯水池への培地の4ミリリットルを加え、貯水池の間に液体の流れを確認してください。 </li></ol></li><li>寒天を作るブリッジ<ol><li> 2インチ長いPVC管（16分の503 ID X 5/16 OD X 1/16ウォール）のペアをカットし、反転し、100ミリリットルのビーカーにそれらを挿入します。 </li><li> 200mgのバクト寒天を測定し、2％寒天ゲルを作るために10ミリリットルの培地で50mlフラスコに追加します。 30秒間電子レンジ。転送ピペットでプラスチックチューブに寒天ゲルをロードします。寒天を固化させる10分間室温で寒天橋のままにしておきます。 </li><li>電気走チャンバの外側のリザーバへの2mlの培地を加える。電流を流すために内側と外側の媒質貯水池に寒天ブリッジを挿入します。 </li></ol></li></ol><img alt="図3" fo:content-width="3.5in" src="/files/ftp_upload/51973/51973fig3highres.jpg" width="350" /> 図3：チャンバーに細胞をロケ電気走室の最終組み立てを実証するための図を。。チャンバは完全に高真空グリースでシールされている。媒体はtで添加されるOリザーバを充填すると、2つの寒天橋がチャンバ内に挿入される。その後、電気走チャンバは顕微鏡ステージに転送され、電極は電界を印加するために取り付けられている。組み立てられた電気走チャンバの側面図は、電流が寒天ブリッジとカバーガラスとの間の空間を通ってセルの上を流れることを実証するために示されている。 3.タイムラプスイメージング<ol><li>前イメージングへの空気循環2時間、5％CO 2で37℃に環境室のスイッチをオンにします。 </li><li>顕微鏡に切り替え、画像取得ソフトウェアを開始する。顕微鏡ステージを校正し、10倍の対物レンズを選択します。 </li><li>顕微鏡ステージに電気走室を移し、テープで室を確保し、細胞に焦点を当てる。右側のカソードと外側の貯水池への電気走電極を挿入します。ワイヤーとテープで電極を固定します。 </li><li>パワーBOに切り替えxは、チャンバに電界を印加する。 25ミリメートル、長室（100 MV / mm）のための2.5ボルトに到達するために、チャンバ全体の電圧計で出力電圧を測定します。出力電流を調整することによって、実験中の電界強度を維持する。 </li><li> 10タイムラプスムービーを生成するソフトウェアでフィルムにチャンバー全体で10ポイントを選択します。 2時間10分間隔で取得条件を設定する。 </li><li>撮影を開始し、必要に応じて出力電流を調整します。 </li><li>実験の最後に、ステージからチャンバを除去し、95％アルコールで細胞を固定する。きれいにカミソリの刃で開放室を破ると、それを再使用しています。 </li></ol>電気走4.定量<ol><li>タイムラプス映画や再オリエント画像のトップにカソードを回転させます。細胞トラッキングソフトウェアに映画をエクスポートします。 </li><li>手動で各映画（ 図4のA、B）から各時点で10〜20細胞の（x、y）の位置を追跡する。南北方向の相対移動距離及び角度は、電界の同じ向きは、細胞追跡ソフトウェア（ 図4C）で計算される。 注：平均移動速度は、総移動距離と総撮影時間から変換された。方向性コサイン値に角度から変換される。細胞は、ランダムな方向性と移行する場合、ネット余弦の平均はゼロに近くなります。細胞は陰極に向かって直接移行すると、余弦値は1になります。細胞は陽極に向かって直接移動する場合は、コサイン値は-1になります。 </li></ol><img alt="図4" fo:content-width="4in" src="/files/ftp_upload/51973/51973fig4highres.jpg" width="400" /> 図4：細胞の画像を追跡線の細胞指向性を測定するために追跡A）オーバーレイ。細胞の（x、y）の位置はmanuaあるLLYタイムラプスムービーで追跡。 。細胞がランダムに移行する場合、平均余弦がゼロに近いB）しかし、細胞はカソードまたはアノードに向かって移動した場合、平均余弦の値が+（カソード）またはに近い- 。（陽極）1.0 C ）方向性は、移行の角度（θ）から変換された余弦値、によって提示されます。コサイン（θ）は、B（電界の方向に投影距離）を遠ざける距離a（移動距離）の比に等しい。 <ol start="3"><li>測定結果を保存します。組み合わせた結果を計算するために、データベース·アプリケーションにデータをインポートします。 </li><li>棒グラフ（ 図5）をプロットするためにスプレッドシートに平均移動速度と平均余弦値の結合されたデータをエクスポートします。 </li></ol>

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R., Inokuchi, J., Tsunoda, T., Lau, A., Ornstein, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+requirements+of+prostatic+epithelial+cell+lines+for+acinar+morphogenesis+and+lumen+formation+in+vitro:+role+of+extracellular+calcium.">Culture requirements of prostatic epithelial cell lines for acinar morphogenesis and lumen formation in vitro: role of extracellular calcium.</a> Prostate. 67 (15), 1601-1613 (2007).</li><li>Lang, S. H., Sharrard, R. M., Stark, M., Villette, J. M., Maitland, N. 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(68), (2012).</li><li>Meng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field-controlled+directed+migration+of+neural+progenitor+cells+in+2D+and+3D+environments.">Electric field-controlled directed migration of neural progenitor cells in 2D and 3D environments.</a> J Vis Exp. (60), (2012).</li><li>Pullar, C. E., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+mediates+keratinocyte+directional+migration+in+an+electric+field.">Cyclic AMP mediates keratinocyte directional migration in an electric field.</a> J Cell Sci. 118 (9), 2023-2034 (2005).</li><li>Sheridan, D. M., Isseroff, R. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle+array+for+measuring+wound+generated+electric+fields.">Microneedle array for measuring wound generated electric fields.</a> Conf Proc IEEE Eng Med Biol Soc. 1, 4326-4328 (2006).</li><li>Nuccitelli, R., Nuccitelli, P., Li, C., Narsing, S., Pariser, D. M., Lui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electric+field+near+human+skin+wounds+declines+with+age+and+provides+a+noninvasive+indicator+of+wound+healing.">The electric field near human skin wounds declines with age and provides a noninvasive indicator of wound healing.</a> Wound Rep. and Reg. 19, 645-655 (2011).</li></ol>

The authors have nothing to disclose.

セルの電気走応答の分析は、多くの細胞の遊走又は増殖27,28を処理するための重要な機能の指標となっている。ここでは二つの前立腺細胞株をフィルムにガラス表面にカスタムメイドのチャンバーを用いる。これらの細胞株は、電気走の異なる程度を実証し、我々は、細胞内局在または電気走媒介タンパク質の活性化は、電気走応答において観察された差が生じる、不死化細胞株を生成するプロセスの間に干渉されるかもしれないと推測している。 培養皿27,28で作ら他の電気走室の設計に比べて、現在の設計にはいくつかの利点がある。まず、ガラス表面は、イメージングのために適しており、細胞を有するカバーガラスを、免疫染色のために電気走後に取り出すことができる。本研究では、セルのグループを監視するために10倍の対物レンズの低倍率を使用する。しかし、THESとeはガラス光学素子の改善には、セル17の前縁にラメリポディアの高速伸縮を追跡する、または蛍光タンパク質で標識した細胞を追跡するために、60倍のオイル対物を有する高倍率での単一細胞を撮影することが可能である。第二に、ガラス表面は、マトリックスタンパク質コーティング10で修飾された12またはマイクロチャネルでパターン化することができる。化学的処理は、29を撮影する前に、細胞を処理する前ことも可能である。マトリックスコーティングは、ここで使用される前立腺細胞株に必要とされず、マトリックスコーティングは、細胞型依存性である。例えば、コラーゲンIコーティングはヒトケラチノサイト30の電気走のために必要であるが、ラミニンコーティングは、ヒト神経幹細胞31の電気走のために必要とされる。我々は、コラーゲンI、コラーゲンIV、フィブロネクチン、およびラミニンにヒトケラチノサイト電気走を試験した。別のマトリックスタンパク質は、細胞遊走の速度を変更したが、正極migratiを変化させなかった<html>電気走29で上。第三に、修正された白金電極は、電場に不活性である。電気走で細胞毒性を減少させる、他の電気走法27で使用されるように銀電極と比較すると、彼らは分解しないか、メディアに金属イオンを放出。第四に、私たちは私たちが高速または低速のどちらか細胞移動を監視するために役立つフレームキャプチャ、間隔を変更することがあります。電動顕微鏡は、一チャンバー内で異なる領域から複数のムービーを獲得する機会を提供する。印加電界なしの対照実験は、電界が細胞の移動速度を変化させるか否かを決定するために重要である。 しかし、現在の電気走チャンバはリザーバ内の媒体の小容量に起因するまで4時間撮影細胞の移動に制限されている。チャンバ設計の拡大版では、より長い期間のために細胞を撮影することが可能である。 <html> ve_content &quot;&gt;方法のいくつかの潜在的な問題が含まれます：。彼らは乾燥になった場合に室（400μL）中の培地の非常に小さなボリュームがあるので1）細胞はチャンバアセンブリの間に死ぬかもしれない、それは空気をトラップ避けることが重要であるチャンバー内の気泡や細胞すすぎ工程の間に細胞をカバーするいくつかの液体を保持する。2）チャンバは、チャンバを通過する電流を減少させるであろう、撮像中に漏れ出す可能性があります。それは前にチャンバ内の液体の流れを注意深くチェックする必要があります顕微鏡ステージに移し、必要に応じて、より高真空グリースを適用した。3）汚れ、高真空グリースが撮影を妨害する可能性がある。ガラス表面はグリースを除去し、95％アルコールで洗浄することができる。4）ステージのドリフトまたは上のチャンバの任意の変位電動顕微鏡ステージは、バイアス方向性のある移動の測定ができた。したがって、適切な段階ホルダーとラベル付けテープで電気走室を確保することが重要である。 ntentは &quot;&gt;全体的に、電気走法はさらに生物における方向の移行を理解するために私たちを助けるもの、印加された電界における運動性の細胞の本質的な方向性を明らかにすることは有用である。

内因性の電界は、皮膚1、32、33、2、脳のような種々の組織で検出される。生理学的な電界が神経突起5,6の伸長を案内し、上皮および角膜創傷閉鎖1,7を促進導く胚発生3,4を含む特定の生物学的機能を提供しています。 インビトロ 、培養細胞に直流電界を印加する模倣生理電界指向性細胞遊走、または電気走を引き起こす。電気走線維芽細胞8、魚ケラチノサイト9、ヒト上皮および角膜ケラチノサイト10-12に検討されている、神経芽2、および神経前駆細胞14,13リンパ球。 （ - ）極印加磁界にさらされたときに、研究された細胞の大部分は、陰極に向かって一方向に移行する。しかし、高転移を含むいくつかの癌細胞ヒト乳癌細胞およびヒト前立腺癌細胞株PC-3Mは、陽極（+）極15、16に移動する。いくつかの機構が電気走を媒介するか、活性化を含む電界を感知する細胞の能力を説明するために提案されているEGFは、12を受容と、上皮ナトリウムチャネル17は 、PI3KおよびPTEN 18、及びカルシウムの放出メカニズムはまだ完全には理解されていない。15、19のイオンとは、複数のシグナル伝達経路が電気走に関与している可能性がある。 ここで実証する2次元電気走法が付着し、運動性細胞の指向性遊走を特徴付けるために、いずれか18、20。この技術は、から変更され、コンフルエント細胞のシートの個々の細胞遊走10、12、17または遊走をモニターするために有用である鵬とJaffe氏21、及び西村らカスタムメイド、透明なPVC室と10、取り外し可能なcoverslとIPSなどの免疫蛍光イメージングなどの二次分析用の電気走、後に簡単にセルの取得を可能にする。電気走室のガラス表面は、高倍率でかつ蛍光標識された細胞での撮影を可能にする、光互換性がある。また、そのような表面コーティングまたは電荷を変化させるように、ガラス表面の改質に実験計画を可能にする。第1カバーガラス製のスペーサーは、細胞上の電流の流れを最小限にするためにチャンバ内で使用されている。したがって、電流の流れの二乗に比例するジュール加熱は、実験中の細胞を過熱しないでしょう。接続寒天ブリッジは細胞と電極との直接接触を防止し、電気走時の培地のpHまたはイオン濃度の変化を防止する。 二つの非腫瘍形成性ヒト前立腺細胞株は、本研究での電気走応答を調べた。 PRNS-1-1 22とPNT2 23は、両方SV4です低または前立腺特異抗原（PSA）の発現なしに上皮マーカーのサイトケラチン5、8、18と19を表す0不死化、増殖因子依存性細胞株。両方の細胞株は、正常な上皮細胞の多角形の形態を維持するが、染色体異常は、染色体分析22、24で観察された。PRNS-1-1とPNT2はほとんどの実験で同様の挙動を共有するが、それらは腺房構造の形成との違いを示して行う電気走。 3-Dマトリックス、マトリゲル上で、PRNS-1-1細胞は、正常な前立腺組織25に類似したルーメンを有する中空腺房構造を形成する。しかし、PNT2細胞が管腔または偏上皮26せずに固体スフェロイドを形成する。 PRNS-1-1細胞はまた、現在の研究でPNT2よりも高いgalvanotactic応答を実証する。 PRNは、1-1腺房構造と電気走の形成の間の相関はgalvanotactic信号が広報を組織において役割を果たし得ることを示唆しているostate腺組織の内因性電界に応答して動き、そして、これら2細胞株とを区別するためにさらなる特性を提供する。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Cells&lt;/strong&gt;</td></tr><tr><td>pRNS-1-1 prostate cells</td><td></td><td></td><td>Lee, &lt;em&gt;et al.&lt;/em&gt; (1994)</td></tr><tr><td>PNT2 prostate cells</td><td>Sigma-Aldrich</td><td>95012613-1VL</td><td>Berthon, &lt;em&gt;et al.&lt;/em&gt; (1995)</td></tr><tr><td colspan="4">&lt;strong&gt;Medium and solutions&lt;/strong&gt;</td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Invitrogen</td><td>11875-093</td><td>warm up to 37 &amp;deg;C before use</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>10% in PBS, warm up to 37 &amp;deg;C before use</td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Life Technologies</td><td>15240</td><td>add 5 ml to 500 ml medium</td></tr><tr><td>2-propanol</td><td>VWR</td><td>BDH1133-5GL</td><td></td></tr><tr><td>PBS</td><td>-</td><td>-</td><td>137 mM NaCl, 2.7 mM KCl, 4.3 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 1.5 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; in 1,000 ml of H&lt;sub&gt;2&lt;/sub&gt;O, pH to 7.4 and autoclaved, warm up to 37 &amp;deg;C before use</td></tr><tr><td>0.25% Trypsin-EDTA&amp;nbsp;</td><td>Invitrogen</td><td>25200-056</td><td>warm up to 37 &amp;deg;C before use, treat cells for 3-5 min at 37 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;Galvanotaxis device&lt;/strong&gt;</td></tr><tr><td>Galvanotaxis chambers</td><td>Precision Plastics Inc, CA</td><td></td><td>Custom-designed (1/4&quot; x 2&quot; x 3.5&quot;), non-toxic, clear PVC chambers. Please contact the authors for the design specs.</td></tr><tr><td>Galvanotaxis electrodes</td><td>UCD electric shop</td><td></td><td>platinum coiled electrodes with flexable cords</td></tr><tr><td>Galvanotaxis power box</td><td>Substrate Engineering, CA</td><td></td><td>custom-designed DC power output with voltmeter</td></tr><tr><td>Microscope Cover Glass, Large, 45 x 50 mm, No. 1.5</td><td>Fisher</td><td>12-544-F</td><td></td></tr><tr><td>Microscope Cover Glass, small, 25 x 25 mm, No. 1</td><td>ThermoScientific</td><td>3307</td><td></td></tr><tr><td>Diamond point marker</td><td>ThermoScientific</td><td>750</td><td></td></tr><tr><td>Marine grade silicon sealer, clear</td><td>3M</td><td>051135-08019</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td>2021846-0807</td><td></td></tr><tr><td>6 ml syringe</td><td>Fisher Scientific</td><td>05-561-64</td><td></td></tr><tr><td>Nichiryo Syringe, 1.5 ml</td><td>Nichiryo</td><td>SG-M</td><td></td></tr><tr><td>Cotton applicators</td><td>Purtian Medical Products</td><td>806-WC</td><td></td></tr><tr><td>Qtips</td><td>Johnson &amp;amp; Johnson</td><td>729389</td><td></td></tr><tr><td>Nalgene 180 PVC tubing&amp;nbsp;</td><td>Nalgene</td><td>8000-9030</td><td>503/16 ID x 5/16 OD x 1/16 Wall</td></tr><tr><td>Bacto-Agar</td><td>Difco</td><td>0140-01</td><td>make 2% agar solution</td></tr><tr><td>Razor Blade</td><td>Personna</td><td>74-0001</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipments and Software&lt;/strong&gt;</td></tr><tr><td>Benchtop Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>operated with an A-4-62 rotor</td></tr><tr><td>Cellometer Auto T4</td><td>Nexcelom</td><td>Auto T4</td><td></td></tr><tr><td>Cellometer counting chambers</td><td>Nexcelom</td><td>CHT4-SD100-002</td><td>load 20 &amp;#956;l cell solutions to count</td></tr><tr><td>Culture Temp Warming plate</td><td>Bel-Art Scienceware</td><td>370150000</td><td>to keep the galvanotaxis chambers at 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Eclipse TE-2000 microscope with motorized stage and environmental chamber</td><td>Nikon</td><td></td><td></td></tr><tr><td>Plan Fluor 10X/0.30 objective len</td><td>Nikon</td><td></td><td></td></tr><tr><td>Retiga EX CCD camera&amp;nbsp;</td><td>Qimaging</td><td></td><td>Cooled CCD camara, mono-color, 12-bit</td></tr><tr><td>Compressed air with 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td></td><td>special order</td></tr><tr><td>Volocity 6.3</td><td>PerkinElmer</td><td></td><td>Image acquiring software</td></tr><tr><td>Improvision OpenLab 5.5.2</td><td>PerkinElmer</td><td></td><td>Cell tracking software and customized to measure migration angles</td></tr><tr><td>FileMaker Pro Advanced, 8.0</td><td>FileMaker</td><td></td><td></td></tr><tr><td>Microsoft Excel 2008 for Mac</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells

生理学的な電場は、胚発生、神経突起成長および上皮創傷治癒における細胞移動を導くような特定の生物学的機能を提供しています。 in vitroで培養細胞に直流電界を印加すると、方向性細胞遊走、または電気走を引き起こす。ここで実証する2次元電気走法は、カスタムメイドのポリ（塩化ビニル）（PVC）チャンバ、ガラス表面、白金電極と細胞が撮像された電動ステージを用いて修飾される。 PVC室と白金電極は、低い細胞毒性を示し、手頃な価格の、再使用可能である。ガラス表面と電動顕微鏡ステージは、画像の品質を改善し、細胞へのガラス表面や治療への可能な修飾を可能にする。我々は2つ​​の非腫瘍形成、SV40不死化前立腺細胞株、PRNS-1-1とPNT2の電気走を撮影。これら2つの細胞株は、同様の移行速度を示し、両方の側に移動カソードが、彼らは電気走における方向性の異なる程度を示します。このプロトコルを使用して得られた結果は、PRNは、1-1 PNT2細胞株は、それらの方向性遊走応答を支配する異なる固有の機能を有することができることを示唆している。

前立腺細胞（PRNS-1-1とPNT2）の2行は、この方法で調査した。両方のライン中の細胞は、2時（ 図5A）の過程で1.0 +/- 0.3ミクロン/分の同じような速度で移動する。しかし、電場の方向性はPNT2ライン（ 図5B）のためPRNS-1-1ラインの0.7 +/- 0.3、および0.2 +/- 0.8である。結果は、それらが位置手がかりに異なる指向性応答を生じる種々の細胞シグナル伝達機構を有していることを示唆し、これら2つの細胞株に（p &lt;0.01、100細胞を追跡した）の電気走の有意差を示す。 <img alt="図5" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig5highres.jpg" width="500" /> 図5：前立腺細胞の電気走。細胞が電界に曝された場合A）PRNS-1-1とPNT2ラインの移動速度が類似している。PNT2ライン電界に低い指向性（コサイン= 0.2）を示しながらB）ただし、PRNS-1-1ラインは、陰極に向かって高指向性（コサイン= 0.7）を示した。

Watch this Scientific Journal Video about Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells at JoVE.com

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

前立腺細胞の方向性のある移動を研究するためにカスタム設計された電気走チェンバースを利用

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

utilizing-custom-designed-galvanotaxis-chambers-to-study-directional

Universidad San Sebastian

Research

JoVE Journal

Biology

9.1K ビュー.  University of California, Davis. We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

ビデオ: Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2Dと3D環境での神経前駆細胞の電界制御監督移行

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

生物工学

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

区分するマイクロ流体デバイスとライブセルイメージングを使用した癌幹細胞の移行の評価

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

医学

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

神経節隆起を培養で生成された細胞の遊走挙動

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

神経科学

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

住セルイメージ投射アッセイ研究神経膠腫脳腫瘍幹細胞の遊走と浸潤に

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

がん研究

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

修正Zigmondチャンバーアッセイを使用してトランク神経冠細胞移動の解析

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

微細加工チャネルにおける細胞移動の研究

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

外部から印加された直流電界における神経前駆細胞の移行動態の解析のための走電性アッセイ

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

3Dコラーゲンマトリックス中の走化性を調査するために、平面グラデーション拡散システム

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS /タクシーと細胞走化性の測定

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

生物学

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

イメージングGタンパク質共役型受容体媒介走化性及び好中球様HL60細胞におけるそのシグナル伝達のイベント

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...