この研究は、韓国国立研究財団(NRF)とハリム大学研究基金の研究助成金(NRF-2018-R1D1A1B07050175、HURF-2017-66)によって支援されました。

ICRマウスを含むすべての研究プロトコルは、元州医科大学延世大学の施設動物管理・使用委員会(IACUC)によって承認されました。実験は世界医師会倫理規定に従って行われた。このプロトコルでは、妊娠中のICRマウスを12/12時間の明暗サイクルに保ち、食物および水に自由にアクセスした。
1. コクリー解剖
<ol><li>紫外線を30分間オンにして層流組織培養フードを殺菌し、使用前に70%エタノールで全ての表面をスプレーします。表面を乾かします。</li>
	<li>解剖器を70%エタノールに10分間入れ、使用する前に乾燥させます。</li>
	<li>手術用ブレードを使用して、出生後3-4日齢のマウスの首を切り落とします(図2A)。</li>
	<li>頭蓋骨を層流フードの実体顕微鏡の下に置き、組織を70%エタノールに浸します。</li>
	<li>組織を組織解剖液(1xハンクスバランス塩溶液、1 mM HEPES)に素早く浸し、エタノールを除去する。</li>
	<li>外科用ブレードで頭蓋骨の中心線を切る(図2B、C)。</li>
	<li>皮膚を前から引き下げ、耳の外耳道を切断して頭蓋骨を露出させる(図2D)。</li>
	<li>眼線を横切って頭蓋骨の前部から後部に切り取る(図2E)。</li>
	<li>頭蓋骨を開き、前脳、小脳、脳幹を鈍い鉗子で取り除く(図2F、G)。</li>
	<li>マイクロ鉗子を用いて、側頭骨から内耳を分離する(図2H)。</li>
	<li>組織解離液を含むペトリ皿にコクリーを移します。</li>
	<li>内の人工内軟組織のみを残して、すべての人工内臓のオティックカプセルを慎重に解剖する(図2I、J)。</li>
	<li>鉗子と別の鉗子のペアで人工内耳管の変化を保持し、ゆっくりと2つの組織を分離する(図2K)。</li>
	<li>ストリア血管症とテクトリアル膜を静かに剥がして取り除く(図2L、M)。</li>
	<li>新しい組織解剖液に殺菌プラスチックカバースリップを入れ、コルティの器官を直径9mmのカバースリップに置き、バジラー膜が下向きであることを確認する(図2N-P)。</li>
	<li>Reissnerの膜と残りの変形組織を鉗子でカバースリップに押し込んで組織を固定化する(図2N-P)。</li>
	<li>埋め込みティッシュのカバースリップを直径35mmの共焦点皿の中心に移す。</li>
	<li>皿の中央に位置する人工内草を持つガラスクローニングシリンダーを皿の上に置き、100 μLの外植培養培地(DMEM/F12、10%胎児血清(FBS)、1%N2サプリメント、アンピシリン(10μg/mL))をシリンダー内に10を加える(図2Q)。</li>
	<li>プレート5×マウス骨髄由来緑色蛍光タンパク質(GFP)タグ付きMSPの10細胞を2mLの培養培地(45%DMEM+ 45%DMEM/F12、10%FBS、1%N2サプリメント、アンピシリン10μg/mL)の外ガラスシリンダー外(図2R)<ol>
<li>MSCが80〜90%コンフルエントである場合、トリプシンエチレンジアミンテトラ酢酸でそれらを取り外すことによってそれらを通過させる。</li>
	</ol>
</li>
	<li>コンフォーカルディッシュを加湿インキュベーターに慎重に移し、5%CO2雰囲気で37°Cで一晩インキュベートします。</li>
	<li>すべての媒体をシリンダーの内側と外側に吸い込み、ガラスシリンダーをコンフォーカル皿から取り外します。</li>
	<li>共焦点皿に2mLの新鮮な培養培地を加え、分析の準備ができるまで加湿インキュベーターで組織培養皿をインキュベートする。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61947/61947fig2v2.jpg"> 図 2.マウスの内臓の解剖とコルチとMSCの器官の共培養.(A)マウスの切断、 (B) および (C) 頭部の中線矢頭解剖、 (D) および (E) 脳の角膜解剖、 (F) および (G) 脳と側頭骨の除去、 (H) 人工内壁の除去、 ( H)骨内壁の除去、 (J)人工内骨の分離、 ( (L) コルチの器官からのストリア血管症 (SV) とスパイラル靭帯 (SL) の分離、 (M) プレート膜の除去、 (N-P) プラスチックカバースリップ上の内骨膜の固定、(Q) コンフォーカルディッシュにおけるカバースリップとガラスシリンダーの位置、 (R) 白スケールバー (A-Ecm = 1)オレンジ(F、G、P)と黄色のスケールバー(H、I)=1mm;緑色のスケールバー(J-O)=0.5 mm.<a href="https://www.jove.com/files/ftp_upload/61947/61947fig2v2large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a> 
2. タイムラプスイメージング
<ol><li>ここで示す実験では、ステージトップインキュベーターシステムを備えた共焦点顕微鏡システムを使用してください。</li>
	<li>共焦点顕微鏡、蛍光灯、コンピュータの電源を入れます。</li>
	<li>共焦点顕微鏡のステージ上に配置したステージトップインキュベーターの条件を37°C、5%CO2雰囲気に設定します。</li>
	<li>皿の固定容器の上にサンプル皿を置き、皿の固定蓋で覆い、上ヒーターの蓋でチャンバーを閉じます。</li>
	<li>視野のコルティとMSCの器官を局地化するためにズームとフォーカスを調整します。</li>
	<li>画像処理ソフトウェアを開きます。 [位置指定 ]オプションで 、20x Plan-Apochromat の目的(0.8 の数値) と 0.5x トリミング領域を選択します。</li>
	<li>[ 取得] で スマート セットアップ をクリックし 、[EGFP]を選択します。</li>
	<li>[取得] の下のチャネルタブを開き、レーザーパワーを0.2% に、ピンホールを44 μmに、マスター ゲインを750 Vに、デジタルゲインを1.0に設定します。</li>
	<li>イメージング設定の下の[ESID]をクリックし、ESIDゲインを4に、デジタルゲインを7.5に設定します。</li>
	<li>210タイルを生成するためにタイルとステークをクリックしてください。</li>
	<li>フォーカス戦略を開き、フォーカスモードを選択します。</li>
	<li>[ 時系列] で、 期間 を 24 時間 、 間隔 を 10 分に設定します。</li>
	<li>[取得] で、フレーム サイズを512 x 512 ピクセル、スキャン速度を8ピクセル、方向を双方向に設定し、平均を4倍に設定し、ピクセルあたりのビット数を16に設定します。</li>
	<li>実験を 開始 するをクリックして実験を開始します。</li>
</ol>3. 画像ファイルの変更
<ol><li>[ 処理] で [ ステッチ] をクリックし、 最小オーバーレイ を 5% に設定し、 最大シフト を 10%に設定します。</li>
	<li>[ ムービーのエクスポート] をクリックし、 非圧縮に設定し、 速度 を 7.5に設定します。</li>
</ol>4. 免疫染色
<ol><li>培地を慎重に吸引し、リン酸緩衝生理食塩分(PBS)でサンプルを5分間洗浄します。</li>
	<li>サンプルをPBSで4%ホルマリンで15分間固定し、サンプルをPBSで3回5分間洗浄します。</li>
	<li>PBSで0.1%トリトンX-100でサンプルを10分間透過し、PBSで3回5分間洗浄します。</li>
	<li>250 μLのファロイジン-iFluor 647試薬(PBSで1:1000希釈)を加え、シェーカーの室温でサンプルを1時間インキュベートします。</li>
	<li>サンプルをPBSで3回5分間洗います。</li>
	<li>カバースリップをガラススライドに移し、2滴の取り付け溶液を追加します。</li>
	<li>スライドにカバースリップをそっと置きます。</li>
	<li>透明なマニキュアでカバースリップを密封し、細胞が観察されるまで暗闇の中で4°Cで保管してください。</li>
	<li>励起/放出(Ex/Em)=650/665 nmのファロイジンおよびEGFPのEx/Em=488/507 nmで、適切なフィルターを備えた共焦点顕微鏡を使用してスライドをイメージします。</li>
</ol>

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著者らは開示するものは何もない。

損傷した細胞の再生を促進するために、損傷部位へのMSCの移植が広範囲に研究されており、治療効果が明らかである。移植とその後のMCの分化は、3-ニトロプロピオン酸13によって誘発される難聴を有するラットの聴力を回復させることが報告されている。Leeらはヒトに対してMSCをトランスベンで適用したが、彼らは聴覚14において有意な改善を達成しなか?...

難聴は先天的に起こり得るか、加齢、薬物、騒音を含むいくつかの要因によって徐々に引き起こされる可能性がある。難聴は、聴力を担う毛細胞が損傷した後に機能障害を回復することは非常に困難であるため、しばしば治療が困難である。世界保健機関(WHO)によると、世界の4億6,100万人が難聴と推定されており、これは世界人口の6.1%を占めています。難聴者のうち93%が成人で、7%が子供です。
多くのアプローチは、難聴を治療するために試みられてきた;特に、MSCを用いた再生アプローチが有望な治療法として登場している。組織が損傷すると、MSCは循環系に自然に放出され、損傷部位に移動し、そこで様々な分子を分泌して再生を促進する微小環境を形成する2。したがって、外部移植されたMSCを標的とする臓器への移行と、強力な免疫調節、血管新生、および抗アポトーシスを引き起こす分子のその後の分泌を通じて損傷した組織を治療する方法を開発することが重要であり、損傷した細胞機能3、4、5の回復を強化する。
MSCが損傷した組織に移行するホーミングプロセスは、克服すべき最も重要な障害である可能性があります。MSCには、テザリング/ローリング、活性化、逮捕、トランスマイグレーション/ダイアペデス、および移行6、7、8の順次ステップを備えた全身ホーミングメカニズムがあります。現在、これらのステップを改善する方法を特定するための取り組みが進行中です。遺伝子組み換え、細胞表面工学、インビトロプライミング、磁気誘導など、さまざまな戦略が6,7で試験されています。さらに、損傷した人工内毛部位にMSCをホーミングすることにより、聴覚毛細胞の保護と再生を促進するためにいくつかの試みがなされている。しかし、インビボでのMSCの追跡は時間と労力を要し、高度に専門的なスキルを必要とします 9.
この問題を解決するために、数時間にわたって細胞の移動を撮影するタイムラプス共焦点顕微鏡を介して、内腔内のMSCのホーミングを観察する方法が開発された(図1)。それは20世紀 初めに開発され、最近特定の細胞の移動を研究するための強力なツールとなっています。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> 図1:図1:(A)コルティの解剖された器官が鉗子を用いてプラスチックカバースリップに付着した後、カバースリップは35mmガラス底の共焦点顕微鏡皿に置かれ、(B)ガラスシリンダーが配置される。(C)ガラスシリンダーの内部を媒体で充填した後、(D)GFPラベル付きMSCを媒体で慎重にシリンダーの外に追加する。(E)一晩のインキュベーション後、ガラスシリンダーを取り外し、画像を共焦点顕微鏡で撮影する。略語: GFP = 緑色蛍光タンパク質;MSC = 間葉系幹細胞。<a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10X PBS Buffer</td>
			<td>GenDEPOT</td>
			<td>P2100-104</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Formalin</td>
			<td>T&#38;I</td>
			<td>BPP-9004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>sigma&#160;</td>
			<td>A5354-10ml</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>sigma&#160;</td>
			<td>A4503-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal dish</td>
			<td>SPL</td>
			<td>200350</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope&#160;</td>
			<td>ZEISS</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>coverslip</td>
			<td>SPL</td>
			<td>20009</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher scientific</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorsheild with DAPI</td>
			<td>sigma&#160;</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Dumont</td>
			<td>0508-L5-P0</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher scientific</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 647 Reagent</td>
			<td>abcam</td>
			<td>ab176759</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage Top Incubator</td>
			<td>TOKAI HIT</td>
			<td>WELSX</td>
			<td></td>
		</tr>
		<tr>
			<td>Strain C57BL/6 mouse messenchymal stem cells with GFP</td>
			<td>cyagen</td>
			<td>MUBMX-01101</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>sigma&#160;</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti

間葉系幹細胞(MCs)が細胞再生および治療に及ぼす影響を調べ、人工内皮との共培養後のMSC移行および形態学的変化を追跡する。コルティの器官は、解剖中に発生したライスナーの膜の一部を押すことによってプラスチックカバースリップに固定化された。ガラスシリンダーで閉じ込められたMSCは、シリンダーが取り外されたときに人工内皮上皮に移行した。彼らの主な局在は、コルチの器官の透析で観察され、神経線維と同様の方向に整列した。しかし、いくつかのMSCは、手足の領域に局在し、水平に細長い形状を示した。また、毛細胞領域への移動が増加し、カナマイシン処理後にMSCの形態が様々な形態に変化した。結論として、この研究の結果は、人工内皮とのMSCの共培養が細胞移植による治療薬の開発および様々な条件および要因を調べることができる細胞再生の研究に有用であることを示している。

3次元モードにおけるMSCのインビトロ移行は、トランスウェルシステムまたは従来の創傷治癒法によって評価され、2次元(2D)モード11での移動を観察する。コルティの器官は、ボエッチャー細胞、クラウディウス細胞、Deiters細胞、ピラー細胞、ヘンセン細胞、外毛細胞、内毛細胞、神経線維、バジル膜、および網状層層などの様々な細胞から構成され?...

Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

本研究では、共焦点顕微鏡を用いて、コルチの器官を含む人工内皮上皮を用いてex vivoインキュベーションによって損傷組織に向かって移動する細胞を観察するリアルタイムイメージング法を提示する。

コルチの器官に向かう細胞の移動を探るインビトロタイムラプスライブセルイメージング

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

in-vitro-time-lapse-live-cell-imaging-to-explore-cell-migration

Research

JoVE Journal

Medicine

3.7K ビュー.  Yonsei University Wonju College of Medicine. 本研究では、共焦点顕微鏡を用いて、コルチの器官を含む人工内皮上皮を用いてex vivoインキュベーションによって損傷組織に向かって移動する細胞を観察するリアルタイムイメージング法を提示する。

ビデオ: In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

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

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

腫瘍の4DライブセルイメージングのためのMAMEモデル：微小環境の相互作用に影響を与える悪性進行

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

前臨床研究における薬理学的チャレンジのfMRIの利用：5-HTシステムへの応用

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

個々の腎細胞がんの中のタンパク質発現変動を探るために逆相タンパク質アレイ（RPPA）の使用

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

歯科用複合材料の細胞適合性評価のための効率的な方法として、共焦点タイムラプスイメージング

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

ボーディッチ影響を調べる：圧力体積関係を測定するために、マウスにおける心臓カテーテル検査

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

LEPR<sup&gt; DB</sup&gt; 2型糖尿病のマウスモデル：無傷の膵島の膵島単離およびライブセル2光子イメージング

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

鏡の国のアリス：単一細胞のタイムラプス顕微鏡と縦トラッキング抗がん治療薬を研究します

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...