Name: fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro
Uploaded: 2018-06-20T12:30:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

著者は、国家自然科学基金、中国の (31371390)、状態のハイテク開発プロジェクト (2014AA020702) のプログラムおよび広東省科学技術 (2016B030231001) のプログラムからの研究助成金のサポートを認めます。

すべての動物の仕事は、動物福祉法、制度のガイドラインに従って実施されますされ、機関動物ケアおよび使用委員会、孫逸仙大学によって承認されました。
1. TEM の準備
<ol><li>頚部転位によってマウスを実行し、滅菌手術用はさみとピンセットを使用して舌を削除します。</li>
	<li>3 分、75% エタノールで舌に没頭し、それぞれ舌を入れて 1.5 mL エッペン (EP) 10 mM 滅菌リン酸 1 mL 管バッファー ソリューション (PBS)。 注: すべての次の手順で PBS の濃度はこの手順で濃度と同じです。</li>
	<li>凍結融解によるセル換散:-80 ° c 1 時間、EP チューブで舌を凍結し、3 回 45 分間室温で舌を解凍します。</li>
	<li>手術針を使用して外科的縫合部分にそれぞれの舌を読み込み、滅菌アルミ箔の小片で縫合糸の端をラップします。無菌状態で 75% のエタノールを含む 3.5 cm や 6 cm の培養皿で操作を実行します。 注: 個々 の手術用縫合糸の適切な長さは約 20 cm、アルミ箔の各部分の適切な大きさは 0.3 cm2 (1 × 0.3 cm)。舌は、アルミ箔に近い読み込む必要があります。</li>
	<li>30 s. 実行無菌状態でこの操作のため 3.5 cm や 6 cm の培養皿で 3 ml の滅菌 PBS の各舌をすすいでください。</li>
	<li>超純水水で舌を洗う: 90 μ g/mL の最終的な集中に滅菌超純水の 250 ml 広口瓶の中にアンピシリンを追加。攪拌棒の入ったボトルに舌を入れてください。残りのボトル外縫合部とボトル キャップを締めます。無菌状態でこの操作を実行します。 注: 縫合の巻きつきを考慮した同じ瓶にで 5 舌までを置くことができます。アルミ箔は、舌をオフに滑りを防ぐためにボトルでは縫合糸の端。舌は、ボトルの内側に残り縫合糸の長さを調整することによって、ボトルの底から 2 センチ配置必要があります。このメモはまた 1.8、1.10、1.12、1.16 の手順です。</li>
	<li>マグネチックスターラー 12 h のためにボトルを置きます。</li>
	<li>塩化ナトリウムと舌を洗う: 滅菌 1 の 250 ml 広口瓶の中にアンピシリンを追加 M 塩化ナトリウム 90 μ g/mL の最終的な集中。舌と攪拌棒をボトルに移動します。残りのボトル外縫合部とボトル キャップを締めます。無菌状態でこの操作を実行します。</li>
	<li>マグネチックスターラー 24 h のためにボトルを置きます。</li>
	<li>セル換散によってトリトン X-100: 滅菌の 2% の 250 ml 広口瓶の中に 90 μ g/mL の最終的な集中にアンピシリンを追加 PBS でトリトン X-100。舌と攪拌棒をボトルに移動します。残りのボトル外縫合部とボトル キャップを締めます。無菌状態でこの操作を実行します。</li>
	<li>マグネチックスターラー 48 h のためにボトルを置きます。</li>
	<li>CaCl2/MgCl2舌を洗う: 90 μ g/mL の最終的な集中に滅菌 5 mM CaCl2/MgCl2の 250 ml 広口瓶の中にアンピシリンを追加。舌と攪拌棒をボトルに移動します。残りのボトル外縫合部とボトル キャップを締めます。無菌状態でこの操作を実行します。</li>
	<li>マグネチックスターラー 24 h のためにボトルを置きます。</li>
	<li>DNase によって消化: ハンクの 1 mL 平衡塩類溶液 (HBSS) 各 EP 管を追加。300 の最終的な集中にそれぞれ HBSS に DNase を追加 μ m 各 EP 管、管の外側の縫合部にそれぞれの舌を移動します。無菌状態でこの操作を実行します。 注: は前の手順でボトル内に残る縫合部がこのステップで EP チューブ内も残ることを確認し、前の手順でボトルの外に残る縫合部がこの手順で EP 管の外側もままかどうかを確認します。</li>
	<li>24 h の 37 ° C で EP チューブで舌を孵化させなさい。</li>
	<li>PBS で舌を洗う: 90 μ g/mL の最終的な集中に滅菌 PBS の 250 ml 広口瓶の中にアンピシリンを追加。舌と攪拌棒をボトルに移動します。残りのボトル外縫合部とボトル キャップを締めます。無菌状態でこの操作を実行します。</li>
	<li>マグネチックスターラー 24 h のためにボトルを置きます。</li>
	<li>使用するまで 4 ° C で滅菌 PBS に準備された TEM を格納します。</li>
</ol>2 TSCC の次元 (3 D) 再構成
<ol><li>
静的 TSCC モデルの構築
	<ol>
<li>種子 1.0 x 106 TSCC 単細胞 (Cal27) 3.5 cm 培養皿に。ダルベッコの 3 mL は、イーグルの中/F12 (DF12) 含む 10% 牛胎児血清 (FBS)、90 μ g/mL アンピシリン 90 μ g/mL カナマイシンを変更を追加します。</li>
		<li>2 〜 3 日の 37 ° C で Cal27 細胞を培養します。セルが少なくとも 60% をカバーすることを確認してください、皿の底のエリア。</li>
		<li>Cal27 単層培養皿の上に TEM をロードします。</li>
		<li>28 日の 37 ° C で CO2インキュベーターに皿を置きます。</li>
		<li>毎日、細胞培養プロセスの中に培養液を更新します。インキュベーターの CO2濃度は 5% です。</li>
	</ol>
</li>
	<li>
攪拌 TSCC モデルの構築
	<ol>
<li>
自作攪拌 minibioreactor の作製
		<ol>
<li>10 mL シリンジのプランジャーを取り出してください。</li>
			<li>ロッドのより低いターミナルのそば (直径 1 cm) 穴を掘り、穴に攪拌棒を読み込みます。</li>
			<li>広口ペットボトルのボトル キャップの中心に穴 (直径 0.5 cm) を掘るし、キャップを介してピストン棒を置きます。</li>
			<li>50 mL の遠心管の半分にカット、ボトル キャップの外側に溶接します。</li>
			<li>釣糸、釣針を結びつけたとロッドをラップすることによって、釣針をロッドに接続します。 注: をロッドで 4 釣針までを接続できます。</li>
			<li>オートクレーブ使用前に、自作の複合体。 注: は、プラスチック製の広口ボトル オートクレーブ滅菌を行います。細胞を培養しながら新しい滅菌プラスチック製の広口ボトルを使用します。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
動的な細胞培養
	<ol>
<li>種子 1.0 x 106 Cal27 単細胞自作 minibioreactor で。10 %fbs、90 μ g/mL アンピシリンと 90 μ g/mL カナマイシンを含む DF12 媒体の 150 mL を追加します。</li>
		<li>TEM をロッドに接続されている釣針を使用して minibioreactor に乗せます。</li>
		<li>ボトル キャップを締めるし、磁性攪拌器に minibioreactor を置きます。7 〜 14 日 37 ° C で CO2インキュベーターで 200 rpm で minibioreactor をアクティブにします。 注: CO2インキュベーターで CO2の濃度は 5% です。</li>
	</ol>
</li>
</ol>

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著者が明らかに何もありません。

脱 ECM 作製の十分に確立されたプロトコル組織で細胞成分を除去しながらネイティブの ECM 成分を保つべきほぼ完全に21。現在報告されている decellularization プロトコル対流輸送によって細胞を除去する血管を介して血流を必要とする、にもかかわらず機械的攪拌ここで採用した、22の伝統的なシンプルで安価な方法として知られています。,<sup class="x...

舌は嚥下、構音、味など、様々 な重要な機能です。したがって、舌機能障害では、患者の生活の質の1が大きな影響を与える。口腔内の最も一般的な悪性腫瘍は舌扁平上皮癌 (TSCC) アルコールを飲酒や喫煙タバコ2人で通常発生します。
近年では、ほとんど進展は、TSCC に関する基礎的研究で達成されています。効率的な体外研究の欠如は、遺跡の最大の問題の 1 つをモデル化します。したがって、細胞外マトリックス (ECM) は、潜在的な解決策になることが判明します。ECM は、高度に組織化されたマトリックス成分から成る複雑なネットワーク フレームは、ECM のような構造と組成を持つ足場材料なので癌研究主務。脱 ECM は、同じ起源の in vitro、ECM の最も重要な利点であることが判明したから細胞の完全にニッチを提供できます。
ECM は、洗剤や酵素を使用して decellularization を組織から削除されている細胞成分を保持ことができます。脱マトリックスのコラーゲン、フィブロネクチン、ラミニンを含む各種の ECM コンポーネントは、生存、増殖、および3細胞の分化を促進、培養細胞のネイティブの組織のような微小環境を提供します。また、移植の免疫原性は、ECM の細胞成分の有無と最低限のレベルに削減できます。
これまでのところ、脱 ECM の作製方法は、さまざまな組織、器官、心臓4,5,6、7、肝8,9,10 などで試されています。 ,11、肺12,13,14,15,16,17、および腎臓18,19,20します。 ただし、関連研究が見つかりません我々 の知識を最大限に舌で同様の作業をします。
本研究では脱舌の細胞外マトリックス (TEM) は一連の物理・化学・酵素治療により効率的にそして安くを作製しました。TEM は、TSCC 動作と開発のための適切なシミュレーションを示す TSCC体外、要約に使用されました。TEM では、TEM TSCC 研究3で大きな可能性があることを示す組織固有のニッチにセルをガイドする機能のほか、良い生体適合性。ここに示すプロトコルは、病因または TSCC の臨床治療研究の選択肢を提供します。

<table><tbody><tr><td>C57-BL/6J mice</td><td>Sun Yat-sen University Laboratory Animal Center</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Guangzhou Chemical Reagent Factory</td><td>HB15-GR-2.5L</td><td></td></tr><tr><td>Sodium chloride</td><td>Sangon Biotech</td><td>A501218</td><td></td></tr><tr><td>Potassium chloride</td><td>Sangon Biotech</td><td>A100395</td><td></td></tr><tr><td>Dibasic Sodium Phosphate</td><td>Guangzhou Chemical Reagent Factory</td><td>BE14-GR-500G</td><td></td></tr><tr><td>Potassium Phosphate Monobasic&amp;nbsp;</td><td>Sangon Biotech</td><td>A501211</td><td></td></tr><tr><td>1.5 mL EP tube</td><td>Axygen</td><td>MCT-150-A</td><td></td></tr><tr><td>Ultra-low temperature freezer&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>3.5 cm cell culture dish</td><td>Thermo Fisher Scientific</td><td>153066</td><td></td></tr><tr><td>6 cm cell culture dish</td><td>Greiner</td><td>628160</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>V900502</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>746495</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>449164</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025</td><td></td></tr><tr><td>Magnesium sulphate</td><td>Sangon Biotech</td><td>A601988</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>158968</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9393</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>PHR1487</td><td></td></tr><tr><td>Surgical suture</td><td>Shanghai Jinhuan</td><td></td><td></td></tr><tr><td>250 mL wide-mouth bottle</td><td>SHUNIU</td><td>1407</td><td></td></tr><tr><td>Magnetic stirrer</td><td>AS ONE</td><td>1-4602-32</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>SHEL LAB</td><td>SCO5A</td><td></td></tr><tr><td>10 mL syringe</td><td>Hunan Pingan</td><td></td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Greiner</td><td>227270</td><td></td></tr><tr><td>Cal27 cell</td><td>Chinese Academy of Science, Shanghai Cell Bank</td><td></td><td>Tongue squamous cell carcinoma cell line</td></tr><tr><td>U2OS cell</td><td>Chinese Academy of Science, Shanghai Cell Bank</td><td></td><td>Human osteosarcoma cell line</td></tr><tr><td>DMEM/F12</td><td>Sigma-Aldrich</td><td>D0547</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P5280</td><td></td></tr><tr><td>Hepes free acid</td><td>BBI</td><td>A600264</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30084.03</td><td></td></tr><tr><td>4 &amp;deg;C fridge</td><td>Haier</td><td></td><td></td></tr><tr><td>Water purifier</td><td>ELGA</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>BLAU</td><td>717805</td><td></td></tr></tbody></table>

fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro

舌扁平上皮細胞癌 (TSCC)の in vitroの効果的かつ現実的なモデルを構築するためにメソッドの作成された食材脱舌細胞外マトリックス (TEM) TSCC 建設のため機能的な足場を提供します。TEM は、細胞の増殖・分化・細胞の生体外でニッチを提供します。ネイティブの細胞外マトリックス (ECM) と decellularized のマトリックスで保持される生化学的組成の微細構造は、セルを固定の組織固有のニッチを提供します。TEM の作製は、深刻な有機または無機の前処理を伴うデオキシリボヌクレアーゼ (DNase) 消化によって実現できます。このプロトコルは操作が簡単ででき、decellularization の高効率。TEM は、TSCC モデルの構築を可能にする静的または撹拌培養条件下で TSCC 細胞の良好な細胞を示した。自作バイオリアクターは細胞培養のための永続的な撹拌条件にも使われました。TEM を使用して再建された TSCC を示した特性と TSCC 病理臨床に似たプロパティ TSCC 研究の可能性を示唆しています。

TEM の準備のためこのプロトコルは効率的かつ適切なをことが判明します。TEM は、母国語の組織と比較して完璧な decellularization を示した。ヘマトキシリン ・ エオジン (HE) 染色 (図 1A B) によって decellularization の有効性を確認した.染色の結果彼は、TEM (図 1B) の染色性が核の完全な消失を明らかにし?...

Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

舌の細胞外マトリックスと舌扁平上皮癌生体外の再構成の作製

効率的な decellularization による舌の細胞外マトリックス (TEM) の準備のためのメソッドを示しています。TEM は、静的または撹拌培養条件下で舌扁平上皮癌 (TSCC) モデルの復興のため、機能的な足場として使用できます。

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

fabrication-tongue-extracellular-matrix-reconstitution-tongue

Research

JoVE Journal

Cancer Research

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

6.2K ビュー.  Sun Yat-sen University. 効率的な decellularization による舌の細胞外マトリックス (TEM) の準備のためのメソッドを示しています。TEM は、静的または撹拌培養条件下で舌扁平上皮癌 (TSCC) モデルの復興のため、機能的な足場として使用できます。

ビデオ: Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

医学

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 ...

がん研究

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

アデノ関連ウイルス-Cas9システムを用いた遺伝子操作マウス頭頸部癌細胞株のインビトロ確立

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

肺扁平上皮癌進行をモデル化する多次元共培養システム

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

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

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

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

癌細胞浸潤への応用による腫瘍間質微小環境の組織工学

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

生物工学

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

3Dオルガノイドによる口腔食道扁平上皮癌のモデリング

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 &#176;C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (&#62;90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

12.5日目および8週齢マウスの舌上皮・間分性/結合組織からの細胞解離

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

発生生物学

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

生物学

Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

Source: Shechtman, L. A. et al. <a href="https://www.jove.com/video/62300" target="_blank">Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells</a>. J. Vis. Exp. (2021)
In this video, we describe the isolation of circumvallate papillae (CVP) epithelium from a murine tongue.

マウスモデルからの環状乳頭(CVP)上皮の単離:マウス舌からCVP上皮を分離するための酵素的手順

Source: Shechtman, L. A. et al. Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells. J. Vis. Exp. (2021)
In this video, ...

JoVE Encyclopedia of Experiments

げっ歯類モデル

成虫

Isolation and Culture of Murine Tongue Epithelial Cells: A Procedure to Isolate and Culture Tongue Epithelial Cells from Mouse Model

Source: Prasad, M. et al. <a href="https://www.jove.com/video/60410" target="_blank">In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line Using an Adeno-Associated Virus-Cas9 System.</a> J. Vis. Exp. (2020)
In this video, we describe the isolation of epithelial cells from a murine tongue. These cells can be further cultured to give rise to cell colonies.

マウス舌上皮細胞の単離と培養:マウスモデルから舌上皮細胞を単離および培養する手順

Source: Prasad, M. et al. In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line Using an Adeno-Associated Virus-Cas9 ...