この作品は、国立科学財団（CBET 0846453）と国立衛生研究所（1R01GM089999）によって部分的にサポートされていました。

1。標準のSU - 8フォトリソグラフィー1（ このビデオでは図示せず ）を用いてシリコンのマスターの作製。 <ol><li>標準のSU - 8クリーンルーム内の異なるコンポーネント（異なる厚さのPDMS膜、共培養チャンバー）製造のためにSU - 8&quot;マスター&quot;（SU - 8 2050、マイクロケム、ニュートン、MA）を作成するためにフォトリソグラフィ法を使用してください。このようなマスターの金型は、任意の微細加工施設（;例えば、スタンフォードマイクロフルイディクス鋳造で作製することができる<a href="http://thebigone.stanford.edu/foundry/">http://thebigone.stanford.edu/foundry/</a> ）。 </li><li>前のPDMSの複製に、マスタからPDMSの簡単なリリースを容易にするために真空源に接続されたデシケーター中でフルオロシラン蒸気にSU - 8のマスターを公開。ペーパータオルにフルオロシランのドロップを追加し、1分間のために真空を適用する。真空を削除し、フルオロシランの堆積のための30分を許可する。 SU - 8今後の使用のために密閉容器に入れてマスターしてください。 </li></ol> 2。マスタ2 SU - 8からPDMSのレプリカ成形<ol><li>流体層と制御層は、SU - 8マスターからPDMSのレプリカ成形によって作られています。 </li><li>午前10時01分重量でPDMSプレポリマーと架橋剤を混ぜる。 1時間（または気泡がPDMSの混合物から除去されるまで）のデシケーター中で比率とドガ。 </li><li>ペトリ皿のSU - 8マスターを置き、慎重にSU - 8のマスターの上にPDMSの混合物を注ぐ。再びドガPDMS。 </li><li>金型で約100μmの均一なPDMSの厚さを得るために1分間1200rpmで金型を回転させる。 </li><li> PDMSを硬化するのに一時間PDMSと80 SU - 8のマスターモールド° Cを含むペトリ皿を加熱する。 </li><li>ホットプレートから硬化PDMS -覆われたSU - 8マスターを削除します。 </li><li> SU - 8マスターからPDMSモールドピール。 </li><li> two細菌の入口、一つの出口一空気圧ポートのために20ゲージの平滑末端針を用いてPDMSモールドの4つの穴を開けます。 </li></ol> 3。多層PDMSデバイスのボンディング：このデバイスが3層、メインチャンネル、中央空気層、および細菌のためのチャネルが含まれている底の細菌層を含む最上位層で組み立てています。下記のように3つの層が組み立てられています。 <ol><li>清潔なピンセットを使用してSU - 8マスター金型からメタノールとし、慎重にはがしPDMS膜を濡らす。テフロンシート上に膜を置きます。 </li><li>酸素プラズマチャンバで空気膜および流体層を配置。 40秒（酸素圧10 SCCM、RFパワー100W）のためにプラズマをオンにします。 </li><li>プラズマへの暴露後、（10分以内）鉗子を用いて底部PDMSチャネル上の膜の位置を合わせます。膜とPDMS層にメタノールを追加すると、レイヤーの自由な移動を支援し、アライメントが容易になります。この場合、デバイスはメタノール蒸気を逃がすためにガーゼに配置されます。 </li><li> 8時間80℃でホットプレート上に整列PDMS層を治す</li><li>結合するために集められた複合PDMSデバイス上に一番上のチャネルを3.4 - 底層に空気の膜を接着した後、手順3.2を繰り返します。 </li></ol> 4。 PDMSデバイス3,4の組み立て<ol><li>真核細胞と細菌を播種するために使用される入口側と出口側の穴を開けます。 </li><li>ポートにタイゴンチューブ（0.01インチOD）を接続し、20mlの注射器で引いて、空気の層を操作する。 </li><li> PDMSモデルと酸素プラズマチャンバ内に事前に洗浄したガラススライドを置きます。ためにプラズマをオンに真空を適用せずに20秒（酸素圧20 SCCM、RFパワー300W）。 </li><li>スライドガラスに島壁（PDMS）ボンディングとの間の接触を防ぐために、すぐにデバイスにシリンジを接続し、シリンジを引いて所定の位置に保持することにより真空を適用する。 </li><li>ゆっくりと（2分以内）ガラス上にPDMSモデルを押してください。 </li><li> 80℃PDMSデバイスを治す℃で10分間を。 </li></ol> 5。フィブロネクチンでガラス表面を処理<ol><li>すべてのポートにタイゴンチューブを接続した後、15分間UVの下でデバイスを滅菌する。 </li><li>チャネル内のいずれかの破片を除去するためにチャネルに滅菌PBSを記入して流れます。気泡を除去するために10分間フローを適用します。空気の泡が簡単にするため、ガス透過性のPDMSをエスケープすることができます。 </li><li>チャンネル内の気泡の形成を防ぐために空気圧のチャンネルに色の色素溶液を埋める。 </li><li>ガラス上に真核細胞の播種を容易にするために、デバイスへのフィブロネクチンの1μg/ mLのソリューションを導入し、37℃で40分間インキュベート℃を</li><li>増殖培地1mLで過剰なフィブロネクチンを洗ってください。 </li></ol> 6。共生E.の形成と測定大腸菌のバイオフィルムの島<ol><li>クローズドチャンバー/島の共生バイオフィルムを形成するためのOD 600までM9最少培地での共生細菌（例えば大腸菌 BW25113/pCM18）〜1に希釈する。 </li><li>正の液圧を印加することにより、島の壁を閉じます。自家製対空液体圧力変換器は、チャネル内の気泡の形成を防ぐために使用されます。 </li><li>共生E.を導入バルブを閉じた状態で島に大腸菌 、および細菌は32℃で1時間のために添付することができます℃に</li><li>アタッチされていない細菌を洗い、12時間0.25mL/hrで希釈細菌懸濁液（OD 600〜0.05）を灌流。 </li><li>緩やかに接続されている細菌を除去し、真核細胞の付着のために準備する新鮮なDMEM培養培地でバイオフィルムを洗浄します。 </li><li>画像ライカTCS SP5共焦点レーザー走査顕微鏡（バノックバーン、イリノイ州）、およびIMARISソフトウェア5を使用してバイオフィルムのアーキテクチャを視覚化を使用してバイオフィルム。 </li></ol> 7。細菌列島6月8日前後真核細胞を播種<ol><li> 5 × 10 5細胞/ mLの播種密度のためにDMEM培地で組織培養フラスコを再懸濁しますからHeLa細胞をトリプシン処理。 </li><li> Picoplusシリンジポンプ（ハーバードApparattus、MA）を用いて2 mL / minの流速で装置にHeLa細胞を導入する。島壁が細菌バイオフィルムは、HeLa細胞領域から独立しているように低下​​していることを確認してください。播種の均一性を決定することができるように、このステップは、光学顕微鏡のステージで実行する必要があります。 </li><li> 5％CO 2インキュベーターで37℃で6時間のためのチャネルとインキュベート° Cで細胞懸濁液の動きを減らすために、セルのコンセントをクランプする。 </li><li>アタッチされていないセルを削除するには、0.5mLの/ hrで増殖培地を使用してデバイスを灌流。 </li><li>リフレッシュHeLa細胞と共生E.ためのDMEM培地大腸菌 6時間ごと。 </li></ol> 8。島と真核細胞への曝露への病原性細菌の導入<ol><li> E.を準備大腸菌 O157：H7（EHEC）は100:1 200:1（HeLa細胞あたりのEHECの数）の感染多重度（MOI）でHeLa細胞に感染する。 </li><li>リンスと緩く接続された共生細菌を洗い流す。 </li><li>島々に希薄EHEC懸濁液（OD 600〜0.1）を導入する。 </li><li> ·フローなしで6時間、5％CO 2インキュベーター内でCが共生バイオフィルム内に存在する信号に公開されるEHECできるようにする37デバイスをインキュベートする。 </li><li> 6時間HeLa細胞に共生の島にEHECを公開するPDMSの壁を持ち上げて。 </li><li> PBSでチャンネルをすすぎ、ライブ/デッドキット（L3224、Invitrogen社、CA）を用いて生細胞と死細胞のために染色。 </li><li>ツァイスAxiovert 200M蛍光顕微鏡での画像の複数の場所をし、生細胞と死細胞の数を見積もります。 </li></ol> 9。代表的な結果9 消化管感染症のイベントのシーケンスは、HeLa細胞の単層と共生E.を開発することによって、模倣された大腸菌のバイオフィルムと前のHeLa細胞の感染に共生バイオフィルムにEHECを公開。マイクロ流体共培養モデルの製造に必要な手順を図1に示します。 図2Aは、共培養装置の3次元レンダリングを示しています。共培養装置真核細胞培養チャンバーと細菌の島の2つの領域が異なる色の染料（細菌の島のための真核生物の領域と緑に紫）と図2Bに示されています。周囲の真核領域から細菌培養領域を単離する可能性は、色の染料を使用して、図2Cに示されています。 図3は、上皮細胞や細菌の共培養から代表的な結果を示す。 図3Aはによって共生バイオフィルム（緑）の島の植民地化を示していますEHEC（赤）。 図3Bは、上皮細胞と共生E.の並置を示しています。細菌の島のEHECと大腸菌 。 EHECとGFP発現共生細菌を表現するRFPは、上皮細胞から細菌の島を単離する可能性を示す、PDMS壁が低下して島にローカライズされています。 <img src="/files/ftp_upload/1749/1749fig1.jpg" alt="図1" /> 図1：共培養モデルにおける細胞播種方式 。 （1）PDMS壁が島を形成するために低下して、共生細菌は、島に導入され、そしてフィブロネクチンは、島の周りに流される。 （2）HeLa細胞は、島を囲む領域に播種されています。 （3）HeLa細胞がコンフルエントに達すると共生バイオフィルムが開発した後は、EHECが島に導入される。 （4）PDMS壁はリフトされていますEHECに島を取り巻くHeLa細胞を公開するまで。バルブ操作の挿入の詳細を示している。 <img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="図2a" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="図2bc" /> 図2：上皮細胞や細菌の共培養のためのマイクロ流体モデル 。上皮細胞間の細菌の島々を形成するための空気圧で作動トラッピング領域を示す共培養装置の（）三次元演出。各細菌の島（1200μmの径と間隔1000μmのは）増殖培地を提供し、島からの廃棄物を除去するための別の入口と出口を持っています。別の地域を（上皮細胞のゾーン、細菌の島々）を示す色の色素と共培養装置の（B）顕微鏡写真。 （C）空気トラッピングシステムの忠実度は、空気圧で活性化チャネル（青）を用いてPDMS壁を（左パネル）下げ閉島の島々に紫色の色素を導入し、48時間、その周囲に黄色の染料を流すことにより示されていますPDMS壁が（右のパネル）が発生したときに、島の地域は、周囲の黄色の染料にさらされている。スケールバーは500μmを表しています。 <img src="/files/ftp_upload/1749/1749fig3.jpg" alt="図3" /> 図3：HeLa細胞と細菌の共培養 。 RFPを発現しているEHECとGFP発現E.の（A）蛍光画像島の大腸菌 BW25113。 （B）送信のオーバーレイ、緑、およびデバイスの赤色蛍光画像。スケールバーは200μmを表しています。

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	<li>McDonald, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prototyping+of+microfluidic+devices+in+poly(dimethylsiloxane)+using+solid-object+printing.">Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.</a> Anal Chem. 74, 1537-1545 (2002).</li><li>Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+integrated+passive+valves+and+pumps+for+flexible+polymer+3-dimensional+microfluidic+systems.">Design and fabrication of integrated passive valves and pumps for flexible polymer 3-dimensional microfluidic systems.</a> Biomed Microdevices. 4, 117-121 (2002).</li><li>Baek, J. Y., Park, J. Y., Ju, J. I., Lee, T. S., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatically+controllable+flexible+and+polymeric+microfluidic+valve+fabricated+via+in+situ+development.">A pneumatically controllable flexible and polymeric microfluidic valve fabricated via in situ development.</a> J Micromech Microeng. 15, 1015-1020 (2005).</li><li>Grover, W. H., Ivester, R. H., Jensen, E. C., Mathies, R. A., A, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+multiplexed+control+of+latching+pneumatic+valves+using+microfluidic+logical+structures.">Development and multiplexed control of latching pneumatic valves using microfluidic logical structures.</a> Lab Chip. 6, 623-631 (2006).</li><li>Lee, J., Jayaraman, A., Wood, T. K., K, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indole+is+an+inter-species+biofilm+signal+mediated+by+SdiA.">Indole is an inter-species biofilm signal mediated by SdiA.</a> BMC Microbiol. 7, 42-42 (2007).</li><li>Hsu, C. H., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+with+tunable+topographies.">Microfluidic devices with tunable topographies.</a> Appl Phys Lett. 86, 023508-023508 (2005).</li><li>Hui, E. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell-cell+interactions.">Micromechanical control of cell-cell interactions.</a> Proc Natl Acad Sci USA. 104, 5722-5726 (2007).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+sensing+hydrogel+microstructures+into+micropatterned+hepatocellular+cocultures.">Integrating sensing hydrogel microstructures into micropatterned hepatocellular cocultures.</a> Langmuir. 25, 3880-3886 (2009).</li><li>Kim, J., Hegde, M., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+epithelial+cells+and+bacteria+for+investigating+host-pathogen+interactions.">Co-culture of epithelial cells and bacteria for investigating host-pathogen interactions.</a> Lab-on-Chip. , (2009).</li></ol>

病原体付着と植民地化のための従来のアッセイは、病原体が追加されるに組織培養プレート中の真核細胞の単層を利用する。これらのモデルは、真核細胞で開発された共生細菌バイオフィルムが内蔵されていないとして、生理学的に適切ではありません。真核細胞への事前成長細菌培養の単純な加算では、細菌の存在下での培養真核細胞に、バイオフィルムは時間をかけて開発する高度に組?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>SU-8 2050</td>
<td>&nbsp;</td>
<td>Microchem Corp, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>high-resolution (16,256 dpi) photolithography mask</td>
<td>&nbsp;</td>
<td>Fineline-Imaging Inc, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>77279</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning, WI</td>
<td>184 SIL ELAST KIT 0.5KG</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH30002.02</td>
<td>&nbsp;</td>
<tr>
<td>Programmable spin coater</td>
<td>&nbsp;</td>
<td>Laurell Tech Corp</td>
<td>WS0650S</td>
<td>&nbsp;</td>
<tr>
<td>Mask aligner</td>
<td>&nbsp;</td>
<td>Neutronix-Quintel, PA</td>
<td>Q4000</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen plasma etcher</td>
<td>&nbsp;</td>
<td>March Plasma System, CA</td>
<td>CS-1701</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Live/Dead Viability/Cytotoxicity Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>L-3224</td>
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microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions

人間の胃腸（GI）管は、腸上皮細胞と非病原性（共生）細菌が共存するユニークな環境です。それは、病原体が共生層に遭遇する微小環境は、植民地化の程度を決定する上で重要であることが提案されている。病原体のコロニー形成を調べるための、現在の培養方法は、よく彼らは消化管の微小環境を模倣する方法で、細菌や上皮細胞の共培養を有効にしていないとしてこの仮説を調べるには適していません。ここでは、真核細胞や細菌の独立した文化を可能にするマイクロ流体共培養モデルを説明し、そして病原体の植民地で共生微環境の効果をテスト。共培養モデルは、共生を開発することにより実証される<em&gt;大腸菌</em腸管出血性の導入に続いて、HeLa細胞の間で&gt;バイオフィルム、<em&gt; E.大腸菌</em&gt;（EHEC）共生の島に、消化管の感染症の一連のイベントを模倣する順序が用いられる。

Watch this Scientific Journal Video about Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions at JoVE.com

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

このプロトコルは、上皮細胞や細菌の同時およびローカライズされた文化のためのマイクロ流体の共培養モデルを説明します。このモデルは、推定されるプロバイオティクス菌株の有効性を病因の異なる水溶性分子シグナルの役割を調べるだけでなく、画面に使用することができます。

マイクロ流体可溶性シグナルを介した相互作用を調査するために上皮細胞や細菌の共培養

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

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JoVE Journal

Biology

14.0K ビュー.  Texas A&M University. このプロトコルは、上皮細胞や細菌の同時およびローカライズされた文化のためのマイクロ流体の共培養モデルを説明します。このモデルは、推定されるプロバイオティクス菌株の有効性を病因の異なる水溶性分子シグナルの役割を調べるだけでなく、画面に使用することができます。

ビデオ: Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

マルチコンパートメントのCNSニューロングリア共培養マイクロ流体プラットフォーム

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

三次元混合細胞スフェロイド共培養から乳腺上皮細胞の分離

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

生物発光共鳴エネルギー転移を用いて生細胞中のタンパク質 - タンパク質相互作用を調査する

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

フローサイトメトリーによる腎上皮細胞における細胞質ゾルカルシウム測定

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

マウス結腸粘膜の顕微解剖のための凍結切片法

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

シングル出芽酵母細胞での寿命と細胞表現型を測定するためのマイクロ流体デバイスを使用しました

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

細菌、酵母、ヒト細胞における一過性形成分子シャペロンアセンブリのモニタリング

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

危険評価のための上皮細胞と原発性免疫細胞からなる多細胞ヒト歯槽モデル

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...