この作品は、ジャクソン州立大学と米国防、アイデア賞 11-1-0205 部環境衛生 RCMI センターを通じて健康の国民の協会 (NIMHD G12MD007581) によって支えられました。

注: このプロトコルは、McArdleらに説明します。11します。図 1のタイムラインを提供します。
1. 培養プレートの準備
<ol><li>ガラス底の疎水性を下げるための 15 分の 1 M 塩酸 (HCl) 1 mL と 10 mm のガラス底を含む 35 mm の培養皿を洗ってください。</li>
	<li>リン酸バッファー生理食塩水 (PBS) すべての HCl を取り除くために 1 mL で 2 回培養プレートを洗います。</li>
	<li>文化の 70% エタノール 1 mL を 2 回洗浄します。</li>
	<li>1 ml の細胞特定の培地の培養プレートをリンス (ここでは、使用 α-MEM 10% 熱を添加した不活性 FBS と 1% 非必須アミノ酸)。 注:次の日まで加湿器では 37 ° C で CO2インキュベーターで培養液を含む処理培養プレートを格納できます。</li>
	<li>35 mm ディッシュの扱われたガラス底に細胞 (MSCs と MCF 7 共培養) を 100% の confluency に成長します。35,000 MSC 細胞および 75,000 24 h MCF7 細胞の共培養します。</li>
</ol>2. コラーゲン ゲルの調製
<ol><li>接触時にコラーゲンの重合を防ぐために冷凍庫にコラーゲン ・ ゲルをピペッティング用マイクロ ピペットのヒントを格納します。 注:これは、最も重要なステップです。</li>
	<li>ボリュームを決定するラット尾コラーゲンのため私はコラーゲン株式の濃度に基づきます。この試金のためのコラーゲン ・ ゲルの 100 μ L を準備します。 注:私は在庫ありラット尾コラーゲンの高濃度が適しています。それに応じてコラーゲンを希釈に使用するダルベッコ変更イーグル培地 x 10 のボリュームを決定します。</li>
	<li>私 (3.8 μ g/mL 在庫)、10 x ダルベッコ変更されたワシの媒体および 1 x 細胞培養液 2% 牛胎児血清と 2.4 mg/mL のコラーゲンの最終的な集中に適切な走化性因子を添加した氷ラット尾コラーゲンを結合します。</li>
	<li>コラーゲン ・ ゲルを中和するために重炭酸ナトリウム溶液の混合物の 4.5% に追加します。 注:重炭酸ナトリウム溶液を追加のボリュームは他の試薬の正確な pH によって異なります。中性の溶液を確保するか、培地の pH 指示薬を使用して pH のストリップとの混合物をテストすることをお勧めします。</li>
	<li>ガラス底培養皿の中で細胞をコラーゲン混合物の 80 μ L を横たわっていた。</li>
	<li>加湿器では 37 ° C で CO2インキュベーターで 30 分間重合コラーゲン ゲルを許可します。</li>
	<li>減らされた血清 (2%) と細胞用培地 2 mL でコラーゲン ・ ゲルをカバーし、24、48、72 時間に加湿器で CO2インキュベーターで 37 ° C でセルを孵化させなさい。 注意:プレートは、ガラス底から切り離す、コラーゲン ・ ゲルを防ぐために慎重に取り扱う必要があります。</li>
</ol>3. コラーゲンの準備の例は、3.8 μ g/mL のラット尾コラーゲン在庫を使用してゲルします。
<ol><li>氷上では、ラット尾コラーゲンの 114.5 μ L、DMEM、x 10 の 16.6 μ L と、走化性因子を含む培地の 33.1 μ L を組み合わせます。</li>
	<li>重炭酸ナトリウム 14.0 μ L でコラーゲン混合物を中和します。</li>
	<li>手順 2.5 のように続けます。</li>
</ol>4. 細胞のイメージング
<ol><li>そっとお皿からメディアを削除します。</li>
	<li>1 ml の PBS のゲルを軽くすすいでください。</li>
	<li>15 分 4% パラホルムアルデヒドの ml の 1 セルを修正します。</li>
	<li>1 mL の PBS で 2 回細胞を洗浄します。</li>
	<li>DAPI 溶液 (100 ng/mL) 室温で 20 分間と細胞を染色します。</li>
	<li>共焦点の顕微鏡を使用して、別の場所での撮影 400 μ m z スタック 16 μ m ステップの各位置のセルをイメージします。顕微鏡の使用により、白色光、アーク励起源を用いた共焦点イメージング ユニットをスキャン ディスクがあります。0.75 の開口で利用されている 20 倍レンズを使用します。</li>
	<li>画像を再構築します。<ol>
<li>与えられたゲル (約 25 画像) の画像を開き、イメージ → スタック → 画像のスタックをクリックして画像のスタックを作成します。ゲルの底に対応した最初のイメージ (z = 0 μ m)、2 番目のイメージは、z 方向の最初のステップに対応した (z = 16 μ m)、z 方向の 2 番目のステップに対応した 3 番目のイメージ (z = 32 μ m)。それぞれのイメージの深さに核を評価し、核はその特定のセルの侵入深さが鮮明で焦点に深さを指定します。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Friedl, P., Brocker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cell+locomotion+within+three-dimensional+extracellular+matric.">The biology of cell locomotion within three-dimensional extracellular matric.</a> Cell Mol Life Sci. 57, 41-64 (2000).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3, 362-374 (2003).</li><li>Yarrow, J. C., Perlman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparison+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Wolf, K., 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=Compensation+mechanism+in+tumor+cell+migration:+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis.">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160, 267-277 (2003).</li><li>Sahai, E., Marshall, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differing+modes+of+tumour+cell+invasion+have+distinct+requirements+for+Rho/ROCK+signalling+and+extracellular+proteolysis.">Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis.</a> Nat Cell Biol. 5, 711-719 (2003).</li><li>Kam, Y., Guess, C., Estrada, L., Weidow, B., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+circular+invasion+assay+mimics+in+vivo+invasive+behavior+of+cancer+cell+lines+and+distinguishes+single-cell+motility+in+vitro.">A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.</a> BMC Cancer. 8, 198 (2008).</li><li>Staton, C. A., Reed, M. W., Brown, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90, 195-221 (2009).</li><li>Poujade, M., 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=Collective+migration+of+an+epithelial+monolayer+in+response+to+a+model+wound.">Collective migration of an epithelial monolayer in response to a model wound.</a> Proc Natl Acad Sci USA. 104, 15988-15993 (2007).</li><li>Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+memory+and+dosing+influence+stem+cell+fate.">Mechanical memory and dosing influence stem cell fate.</a> Nat Materials. 13, 645-652 (2014).</li></ol>

著者は、競合する関心が存在しないことを宣言しています。

ここでは、様々 な細胞などの微小環境に依存したり、まれにあるセルに便利です簡単な反転垂直浸潤能の測定法について述べる.この 3 D 浸潤アッセイの細胞は彼らの元の微小環境の維持し、走化性因子を含むコラーゲン ゲルで覆われています。セルは移行し、コラーゲンに侵入します。このアッセイで細胞を染色し、ゲル内で簡単に監視できます。シンプルさとこのアッセイの再現性は、3 ...

細胞運動は細胞の正常発達的および生理学的プロセスです。ただし、パターンと移行し、侵入する細胞の能力の変化は、炎症性疾患、がん転移1,2,3などの病態に関連付けられます。転移がんで最も悪い理解の側面といえます。転移、がん細胞は細胞外マトリックス (ECM) が低下する、ローカル組織および intravasate に侵入する必要があります。循環では、がん細胞は、遠くのサイトに発信する、extravasate、二次性腫瘍を形成します。したがって、能力 ECM を低下させる細胞の移行して侵入するが、転移能に関連します。異なるアプローチは、分析、細胞の移行と侵略の機能を定量化して開発されています。最もよく使われる方法には、創傷治癒の試金、微速度顕微鏡観察、ボイデン室アッセイが含まれます。創傷治癒の試金4と時間経過顕微鏡検査、細胞遊走に予備的な洞察力を与えるだろう簡単で便利な試金。ただし、細胞が生体内で存在する 3 D 環境を反映して 2 D の試金はどちらも。研究では、細胞が 2 D 15,6と比較して 3 D 環境に異なる反応することを示しています。さらに、創傷治癒の試金は再現し、量的な結果7,8,9をもたらす困難です。創傷治癒のアッセイの 3 D の変更、セルの除外ゾーンの試金はもっと生理学的に関連するアッセイが細胞融合イベント10,11 から生じるハイブリッド細胞など数の少ないセルに適していません.
ボイデン室アッセイは、細胞研究関連微小を提供する 3 次元試金です。ただし、セルの元の微小環境から削除して移行し、走化性因子含む培地に向かって下方に侵入・ ボイデン ・ アッセイ系の上部室に預けられるが必要です。この 3 D のアプローチは細胞の微小環境やで限定数10、11,12に脆弱性の移行および侵略の機能の分析に適していません。細胞の遊走・浸潤を分析する新しいアプローチは、マイクロ流路勾配商工会議所アッセイ13です。適した、移行および侵略の研究の信頼できる、この試金はより高価な典型的な実験室の技術的に困難な場合が。ここで、微小環境に敏感な細胞を含む多くの細胞の種類と互換性があるおよび/または数の制限は、単純な反転垂直浸潤アッセイについて述べる。この試金はフーパーらによって提案されたプロトコルから適応されました。14します。 この分析では、セルのまま彼らの元の環境での移行と侵略が走化性因子の11を含むコラーゲン ・ ゲル内上方に移動ことを監視します。この試金は再現はされて最適化し、35 mm ディッシュで検証します。それは、異なるセル文化のサイズ、異なるマトリックス付着、および異なる塩基の強さなどさまざまな分析条件に適応できます。この試金は、創薬プロセスの初期スクリーニングのための in vitroプラットフォームとして役立つことができます。

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="880">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">MatTek P35G-1.5-10C</td>
			<td style="width:269px;">MatTek Corporation, Ashland, MA</td>
			<td style="width:151px;">P35G-1.5-10-C</td>
			<td style="width:232px;">mattek glass bottom dishes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat tail collagen I</td>
			<td>Corning, Corning, NY, USA</td>
			<td align="right">354236</td>
			<td>Collagen gel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>H1758</td>
			<td>HCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">10010023</td>
			<td>PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>D2429</td>
			<td>10X DMEM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>S5761</td>
			<td>NaHCO3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Fluorescent microscope</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>Olympus IX81</td>
			<td>Confocal&#160; microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defined Fetal Bovine Serum</td>
			<td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td>
			<td>SH30070.03</td>
			<td>FBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ti:Sapphire multi-photon laser scanning microscope</td>
			<td>Prarie Technologies, Sioux Falls, SD, USA</td>
			<td>40x NA 0.8 objective</td>
			<td>MPLSM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris software</td>
			<td>Bitplane Inc. Zurich, CH</td>
			<td>Imaris 7.5.2</td>
			<td>Imaris software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td style="width:269px;">Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>D9542</td>
			<td>DAPI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">50980487</td>
			<td>PFA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#945;-minimum essential medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>M0894</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MDA-MB-231</td>
			<td>ATCC; Manassas, VA, USA</td>
			<td>HBT-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MSC</td>
			<td>Trivedi and Hematti, 2007</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20X lens UPLAPO</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>36-064</td>
			<td>Olympus UPLSAPO 20X Objective</td>
		</tr>
	</tbody>
</table>

moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol

3 D 浸潤アッセイを開発しているが、その微小環境の整合性に影響を与えずに細胞を研究課題が残っています。ボイデン室必要セルが元の文化場所から避難して、新しい環境に移動するなど、従来の 3次元の試金します。だけでなく、これが、微小環境に固有な細胞プロセスを混乱させる、それは多くの場合細胞の損失の結果します。まれ、かの微小環境に非常に敏感な細胞を扱うとき、これらの問題は特に挑戦的です。ここでは、両方の問題を回避する 3 D 浸潤能の測定法の開発について述べる.この分析では、小規模でも細胞をめっきし、走化性因子を含む ECM 行列をセルの上に置いた。これはセル変位を必要としない、上向きマトリックスへの侵入セルをできます。このアッセイでは、細胞の形態と同様、細胞浸潤は、コラーゲン ・ ゲル内で評価できます。この試金を使用して、我々 は稀で、敏感な細胞の侵襲的な容量を特徴付ける乳癌細胞 MCF7 と間葉系/多能性の融合から生じるハイブリッド細胞幹/間質細胞 (MSCs)。

ガラス下部およびラット尾コラーゲン入り 35 mm 培養皿を用いて開発し、生体外で3 D 侵略を最適化する私はプロトコルを試金。我々 は癌細胞 MCF7 と MSC 胸を示したこのアッセイを使用して細胞が 0.04 ± 1.8 μ m、μ m ± 76.0 41.3 平均距離にコラーゲン ・ ゲルに 48 時間後それぞれ侵入しとき IGF1 (18.6 ng/mL) は、走化性因子 (図 2) として使用され?...

Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

このプロトコルでは、細胞の微小環境を維持しながら三次元の設定のセルの移行と侵略の機能を定量化に使用できる反転垂直浸潤能の測定法について説明します。この試金は稀および/または敏感な細胞に適しています。

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

moving-upwards-simple-flexible-vitro-three-dimensional-invasion-assay

Research

JoVE Journal

Cancer Research

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

6.0K ビュー.  University of Minnesota - Twin Cities. このプロトコルでは、細胞の微小環境を維持しながら三次元の設定のセルの移行と侵略の機能を定量化に使用できる反転垂直浸潤能の測定法について説明します。この試金は稀および/または敏感な細胞に適しています。

ビデオ: Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

創薬スクリーニングの高スループット三次元腫瘍回転楕円体の生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

がん研究

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

CD8 による培養腫瘍細胞のアポトーシスのリアルタイム検出+ T 細胞腫瘍浸潤骨髄細胞の免疫抑制機能を勉強するには

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

自動生細胞固体撮像素子を使用して単純な移行/侵略ワークフロー

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉腫に対する三次元骨細胞外マトリックス モデル

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

三次元スフェロイド浸潤アッセイにおける完全ヒト腫瘍ベースマトリクス

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

腫瘍マクロファージ相互作用を研究するインビトロアッセイ

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

がん関連線維芽細胞分化とインビトロ研究における侵入のためのより生理学的システムとしての3Dスフェロイドモデル

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

患者前立腺癌骨転移標本及びその異種移植片に由来する3次元(3D)オルガノイドの確立と解析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

大腸癌幹細胞を研究するスフェロイドの3次元モデル

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

コラーゲンベースの足場を用いた神経芽細胞腫の3次元 in vitro 生体模倣モデル

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

要約

さらに動画を探す

この動画の章

創薬スクリーニングの高スループット三次元腫瘍回転楕円体の生成

CD8 による培養腫瘍細胞のアポトーシスのリアルタイム検出⁺ T 細胞腫瘍浸潤骨髄細胞の免疫抑制機能を勉強するには

自動生細胞固体撮像素子を使用して単純な移行/侵略ワークフロー

骨肉腫に対する三次元骨細胞外マトリックスモデル

三次元スフェロイド浸潤アッセイにおける完全ヒト腫瘍ベースマトリクス

腫瘍マクロファージ相互作用を研究するインビトロアッセイ

がん関連線維芽細胞分化とインビトロ研究における侵入のためのより生理学的システムとしての3Dスフェロイドモデル

患者前立腺癌骨転移標本及びその異種移植片に由来する3次元(3D)オルガノイドの確立と解析

大腸癌幹細胞を研究するスフェロイドの3次元モデル

コラーゲンベースの足場を用いた神経芽細胞腫の3次元 in vitro 生体模倣モデル

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

要約

さらに動画を探す

この動画の章

創薬スクリーニングの高スループット三次元腫瘍回転楕円体の生成

CD8 による培養腫瘍細胞のアポトーシスのリアルタイム検出+ T 細胞腫瘍浸潤骨髄細胞の免疫抑制機能を勉強するには

自動生細胞固体撮像素子を使用して単純な移行/侵略ワークフロー

骨肉腫に対する三次元骨細胞外マトリックス モデル

三次元スフェロイド浸潤アッセイにおける完全ヒト腫瘍ベースマトリクス

腫瘍マクロファージ相互作用を研究するインビトロアッセイ

がん関連線維芽細胞分化とインビトロ研究における侵入のためのより生理学的システムとしての3Dスフェロイドモデル

患者前立腺癌骨転移標本及びその異種移植片に由来する3次元(3D)オルガノイドの確立と解析

大腸癌幹細胞を研究するスフェロイドの3次元モデル

コラーゲンベースの足場を用いた神経芽細胞腫の3次元 in vitro 生体模倣モデル

CD8 による培養腫瘍細胞のアポトーシスのリアルタイム検出⁺ T 細胞腫瘍浸潤骨髄細胞の免疫抑制機能を勉強するには

骨肉腫に対する三次元骨細胞外マトリックスモデル