Name: exploring the potential of mesenchymal stem cell sheet on the development of hepatocellular carcinoma in vivo
Uploaded: 2018-09-11T12:30:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo at JoVE.com

著者は、彼らの協力とサポート特にフセイン Almukhayzim と Hisham Aloudah 実験的手術と動物キング サウド大学医学研究所のスタッフに感謝したいと思います。著者はまた視覚を準備するためキング サウド bin Abdul-Aziz 大学でメディア チームを認めるたいと思います特に素材 Muath ビンガーンナムと Abdulwahab Alsulami。

プロトコルは、キング サウド大学倫理委員会の動物のケアのガイドラインに従います。動物で使用される他の薬、麻酔薬、外科的処置は、キング サウド大学倫理委員会で承認されます。すべての作品は、適切に訓練された人員によって実行されます。
1. 細胞シート工法
<ol><li>原液ウシ胎児血清 (FBS) で培養皿 (3.5 cm 温度応答性培養皿) をコートし、料理のすべての面をカバーすることを確認します。 注: この手順は、単層の細胞の成長をサポートし、細胞凝集を防ぐことができますので細胞シートの剥離のために重要です。</li>
	<li>5% CO2と 95% 空気の入った加湿雰囲気で 37 ° C で 24 時間の料理を孵化させなさい。</li>
	<li>コーティングの料理から余分な FBS を削除し、少なくとも 1 時間 (RT) 室温で乾燥する料理を許可します。</li>
	<li>3 つの細胞懸濁液を準備: 1 x 106 HepG2 細胞、BMMSCs、1 x 106 HepG2 細胞および UCMSCs (BMMSCs と UCMSCs の両方の 4:1 の比率) で 1 × 106 HepG2 細胞。</li>
	<li>それぞれのラベルの付いた皿に適切な細胞懸濁液をシードします。</li>
	<li>10 %fbs と 1% ペニシリン/ストレプトマイシン添加 DMEM 培養液中のすべてのセルを停止します。</li>
	<li>37 ° c 5% CO2と 95% 空気の入った加湿雰囲気でセルを孵化させなさい。</li>
</ol>2. 細胞シートの剥離
<ol><li>100% セル合流、37 ° C の定温器から料理を取る。</li>
	<li>HepG2/BMMSC と 5% CO2と 95% 空気の入った加湿雰囲気で 20 ° C で 30 ~ 40 分の HepG2/UCMSC 細胞シート料理を抱卵中の常温では、1 h の HepG2 細胞シート皿を孵化させなさい。 注: hepg2 細胞のみから作った細胞シートは壊れやすいです。シートの構造への損傷を引き起こす 20 ° C に温度を下げます。</li>
</ol>3. 手術の手順のパフォーマンス
注: 細胞シートの剥離の培養時間の間に、動物のプロシージャを開始します。
<ol><li>
外科領域の準備について
	<ol>
<li>フード、キャビネット、層流無菌手術室など、研究室内滅菌の別の領域ですべての手術を行います。外科領域のすべてのサーフェスが非多孔性、密封された、耐久、そして墨であることを確認します。</li>
		<li>ボディス クラブ、手袋、マスクをつけた、頭と靴カバーなど、適切な保護具を着用します。</li>
		<li>手術使用ツールは清潔で滅菌 (オートクレーブ経由; 高圧飽和幹) 初めに手術。</li>
		<li>ラット間の手袋を変更します。</li>
	</ol>
</li>
	<li>
動物の手術のための準備
	<ol>
<li>8-12 週齢裸ラットを使用します。</li>
		<li>注射麻酔ラットを作成します。<ol>
<li>麻酔ソリューションを準備するには、ケタミン ・ キシラジン、ケタミンの 2 mL、キシラジン (20 mg/mL の濃度) での 1 mL、1 mL の滅菌生理食塩水 (例えば0.9% 塩化ナトリウム) 滅菌 10 mL の血清コレクション バイアルをミックスし、前によく振って使用します。</li>
			<li>麻酔を適用するには、ラットの体重 100 g 当たりケタミン ・ キシラジン溶液 0.2 mL を腹腔内注入します。 注: ソリューションの総投与量は 100 mg/kg のケタミン ・ キシラジンの 10 mg/kg です。</li>
		</ol>
</li>
		<li>手術部位から目に見える汚れやごみを削除します。</li>
		<li>ペダルの逃避反射 (両方の後部のフィートの足パッドのピンチ) をテストすることによって麻酔の深さを確認します。 注: 足パッドのピンチには、応答が発生した場合 0.05 mL/100 g (約は 30 分毎)、用量で繰り返し、麻酔の深さを再確認します。</li>
		<li>ポビドン ヨード/イソプロパノールして 2 段スクラブで切開部を消毒します。</li>
		<li>切開の汚染を避けるために滅菌ドレープでラットをカバーします。</li>
		<li>ブプレノルフィン (0.01 - 0.05 mg/kg) とラットの皮下挿入8切開をする前に。</li>
		<li>生殖不能のメスを使用して、7-に 10 センチ皮と肝を公開するための基礎となる筋肉のカットを作る。<ol>
<li>メスのフラット バックの端と皮膚から腹部の筋肉を分離します。</li>
			<li>慎重に刺して、 linea albaメスを使用して鋭いハサミでカットを拡張します。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 細胞シート移植
メモ: 肝臓の細胞シート移植は実行されます。
<ol><li>培養皿の細胞シートを収集します。<ol>
<li>その面からシートをデタッチするベンチの上培養皿を軽くたたきます。</li>
		<li>滅菌ピペット チップを使用して半円形でシートの端を削って、料理の面から細胞シートを区切ります。</li>
		<li>優しく培養皿からすべてのメディアを削除します。</li>
		<li>シートが損傷; せずそのままを確認します。そうでなければ、することはできません (図 1) を使用します。 注: 結果のシートは、目で直接検出できます。</li>
	</ol>
</li>
	<li>お皿から細胞シートを収穫する無菌膜 (ろ紙) を準備します。<ol>
<li>まず、通常の生理食塩水を膜を浸します。滅菌綿パッドで過剰な生理食塩水を削除します。</li>
		<li>細胞シートにシワや膜と細胞シートの間の空気の泡を回避しながらウェット膜を置きます。</li>
		<li>膜とそれらを簡単に収集する細胞シートを食塩の数滴を追加します。 注: 細胞シートを破損しないよう、冷たい生理食塩水を使用し、ない鉗子で膜と細胞シートを持ち上げます。</li>
		<li>細胞シートは、膜に正しく接続されていることを確認するため、数秒間の膜を残すし、それを収集します。</li>
	</ol>
</li>
	<li>細胞シート移植肝を準備します。<ol>
<li>滅菌コットンでやさしくタッピングによって肝臓の表面が乾燥していることを確認します。</li>
		<li>滅菌鉗子を使用すると、細胞シートの膜をラットの肝の 1 つの葉にかぶせます。</li>
		<li>滅菌生理食塩水を数滴を追加し、そこから細胞シートをデタッチする膜に穏やかな圧力を適用します。</li>
		<li>慎重に膜を削除する前に 5 分待ちます。 注: 細胞シートは肝臓に正しく接続されていることを確認するこれは。</li>
		<li>最後に、基になる筋肉を閉じるし、5-0 ナイロン縫合糸で切開を皮膚します。</li>
		<li>ポビドン ヨードと手術領域を消毒します。 注:図 2は裸のラットの肝臓細胞シート移植手順の図を示します。</li>
	</ol>
</li>
</ol>5. 手術後のケア
<ol><li>手術後すぐに共食いしたり、窒息を避けるために個別にラットの家し、それが定期的に (少なくとも 15 分) を監視する意識であり、完全に外来まで。</li>
	<li>その後、合併症がないことを確認する, 5-14 d、5 d、少なくとも毎日のラットを評価します。毎日の評価中に、次のことを確認します。<ol>
<li>傷口が閉じていると、縫合糸がきつすぎることがなく、そのまま。</li>
		<li>過剰な熱などの切開部位に感染の兆候がない、腫れや膿の排出。</li>
		<li>食料や水の消費を落ち込ませる、体重減少、脱水、皮膚テンティング、迅速かつ開いて口呼吸などの痛みに関連付けられている兆候はありません。手術後疼痛に適度な制御にあれば、注入ラット皮下ブプレノルフィン (0.01 - 0.05 mg/kg) と 48-72 時間の最小値のためのすべての 6-8 h9術後。</li>
	</ol>
</li>
</ol>6. 移植領域の解析
<ol><li>Scarifice ラット移植後 1 ヶ月の移植領域を収集し、組織学的および免疫組織化学的解析。</li>
</ol>

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著者が明らかに何もありません。

研究の豊富な量は、モデルの開発生体内で十分な前臨床動物人間癌のように取り組んでいます。現在、癌の動物モデルを作成するために使用の主要なアプローチは、遺伝子工学や細胞移植11を含みます。遺伝子組み換え動物モデルは、同定と薬剤誘発性毒性発現の分子機構を理解することと同様、ターゲット遺伝子の検証のための良いツールです。しかし、このモデ?...

肝細胞癌は予後に有意関連付けられている肝臓の癌です。毎年、ほぼ 50万人新規患者は、肝細胞癌、肝細胞癌患者世界1の 85% を表すと診断されます。発癌ではない単一フォーム病気;むしろ、それはコレクションに加え別の病理組織学的特徴と遺伝子およびゲノム変動している病気の予後の結果1を様々 なです。したがって、肝細胞癌に対する有効な治療戦略の開発の主な課題は、肝細胞癌の生物学の限られた知識とこの複雑な病気の理解を助けることができる適切な実験動物モデルの欠如です。ひと癌を模倣した生体内で動物モデルは候補者の遺伝子を選択し、関与する癌の応答に影響を及ぼすさまざまな要因を調査するためだけでなく、癌を誘発、予後/予測マーカーを識別するために必要です治療上のエージェント。
In vitro癌研究はまだ主要な制限に関連付けられます。これは、がん細胞は文化で維持されるとき体内の機能の多くを失うためにです。前のヴィヴォ設定全体組織生理学の不在から細胞培養結果に発生する変更。がん細胞間相互作用(上皮間質、免疫、血管系等)内腫瘍微小環境はがん細胞の特性2に大きく反映されます。がん微小環境はがん細胞遺伝子・ タンパク質発現と血管新生に対する抑制と転移ポテンシャルに加えての表現型特性を変えることができます。文化システムの in vitro二次元 (2 D) は、腫瘍の進行を調節するために必要な適切な組織のマトリックスを欠いています。したがって、これらの制限のためは、 in vitroモデルの予備調査結果をサポートする体内モデルを利用する必要がありますは常にあります。この研究では、細胞シート技術を使用して、HCC の基になる完全な生物学的プロセスを解明する生体内で動物モデルを開発します。
10 年以上前、岡野の研究所は、細胞シート技術3に基づく組織工学の手法を設立しました。この手法は、表層の疎水性を制御することにより可逆的な細胞接着/脱着できるように熱応答性培養プラスチックを採用しています。このメソッドでは、手入れの行き届いた細胞外マトリックス (ECM) と細胞間相互作用に、そのまま立体 (3 D) 形式 (i.e.,cell 枚)、培養細胞の穏やかな収穫ができます。細胞シート技術は、商業的に使用可能である (ピパよ)、温度応答性高分子 poly(N-isopropylacrylamide) の培養皿のプレコートを必要があります。20 ° C の下の温度、ピパよポリマー水和し、高い温度 (37 ° C) でポリマーが脱水状態になるし、濁水沈殿物に変換に対し、水溶液に溶解します。ポリマーは親水性のアミド チェーンと疎水性側鎖 (イソプロピル グループ) に含まれます。一方、水和構造の内訳のイソプロピル グループと疎水性イソプロピルのグループを囲む水の分子を集約低温で高温で水の分子のブラウン運動が激化は疎水性相互作用のためしたがって、ポリマーの全体の鎖は集計し、4を析出します。
本研究では 3 つの異なる細胞シートを用いた肝細胞癌の動物モデルの開発にこの手法を採用します。採用されている最初のシートから成って肝細胞のみ、他の 2 つのシートから成っている肝細胞と 2 種類のソースからの MSCs の組み合わせに対し: 骨髄 MSCs (BMMSCs) と臍帯 MSCs (UCMSCs)。MSCs は、非造血細胞脂肪細胞、骨細胞、軟骨細胞、筋5を含む、間葉系の系統の intocell 誘導体を区別することができます。これらの細胞を用いてがん細胞シートを作成するときの理由は、癌に及ぼす MSCs 矛盾したレポートです。MSCs が 2 つの異なる表現型を持つことが示唆されている:"MSC1"、炎症性表現型と「MSC2」、免疫表現型6。MSCs は、toll 様受容体 (Tlr) を表現します。MSCs の TLR4 プライミングは、TLR3 プライミング6免疫抑制因子の分泌を増加させるに対し、炎症性因子の分泌を増加します。これらの 2 つの表現型の体外研究報告 MSC2 共同文化があった反対の効果7MSC1 の癌細胞との共培養減衰、がん細胞の成長であること。これは、MSCs はプロがんや抗がん剤、その表現型によってできることを関係づけます。したがって、細胞シート技術を用いた肝細胞癌の動物モデルの開発に加えたい MSC 移植の腫瘍の開発に及ぼす影響を探索するかどうかこれらの細胞を使用して高めるか、または減らすこのモデルの開発と。

<table><tbody><tr><td>&lt;strong&gt;Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Gibco/Invitrogen</td><td>10270106</td><td></td></tr><tr><td>DMEM high glucose&amp;nbsp;</td><td>Sigma</td><td>D5671-500ML</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Life Technology</td><td>15070063</td><td></td></tr><tr><td>Sterile physiologic saline</td><td>Sigma&amp;nbsp;</td><td>S0817-1GA</td><td></td></tr><tr><td>Human HepG2 cell line</td><td>ATCC, USA</td><td>HB-8065</td><td></td></tr><tr><td>Human bone marrow MSCs cell line</td><td>PromoCell, USA</td><td>C-12974</td><td></td></tr><tr><td>human umbilical cord tissue MSCs</td><td>PromoCell, USA&amp;nbsp;</td><td>C-12971</td><td></td></tr><tr><td>Ketamine 50%</td><td>Rompun, Bayer</td><td></td><td></td></tr><tr><td>Xylazine 2%</td><td>Rompun</td><td>23076-35-9</td><td></td></tr><tr><td>Alphadine&amp;reg;&amp;nbsp;solution.</td><td>Riyadh Pharma</td><td>LBL0816</td><td></td></tr><tr><td>&lt;strong&gt;Disposables:&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15mL Polypropylene High Clarity PP Centrifuge Tube</td><td>Falcon&amp;nbsp;</td><td>352097</td><td></td></tr><tr><td>3.5 cm sterile UpCell culture dishes with the filter paper (membrane)</td><td>Sigma</td><td>174904-1CS</td><td></td></tr><tr><td>100-1000 &amp;micro;l &amp;nbsp;Pipette Tips&amp;nbsp;</td><td>Sigma</td><td>CLS4868-1000EA&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Basic Procedure Drape</td><td>Thermofisher</td><td>PMD5293.0</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plus pipette, variable volume</td><td>Eppendorf&amp;reg;&amp;nbsp;Research&amp;reg;</td><td>Z683779-1EA</td><td></td></tr><tr><td>Tissue culture incubator 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Any brand</td><td></td><td></td></tr><tr><td>Biological safety cabinet</td><td>Any brand</td><td></td><td></td></tr><tr><td>Tissue culture incubator 20 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Any brand</td><td></td><td></td></tr><tr><td>Sterile surgical tools and nude rats:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>scalpel&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Nylon Suture&amp;nbsp; 5-0</td><td>Accutome</td><td>AB-3854S</td><td>Monofilament, Lancet</td></tr><tr><td>1 ml Tuberculin Syringes</td><td>Fisher Scientific</td><td>14-826-88</td><td></td></tr><tr><td>Nude rats&amp;nbsp;</td><td>Charles river</td><td></td><td></td></tr></tbody></table>

exploring the potential of mesenchymal stem cell sheet on the development of hepatocellular carcinoma in vivo

ひと癌を模倣した生体内で動物モデルは、重要な臨床情報を提供するさまざまなアプリケーション可能性があります。生体内で癌モデルの開発に現在使用されている技法には、かなり制限があります。したがって、本研究では生体内で癌モデルを開発する細胞シート技術の実装を目指します。肝細胞癌 (HCC) は肝細胞から作成した細胞シートを用いた裸のラットの開発に成功。細胞接着、細胞外マトリックスによって制御される、層状構造の形成を介して癌細胞シートが生成されます。HCC シート移植肝臓に月内担癌動物モデルの作成が可能になります。さらに、この癌モデルの開発の間葉系幹細胞 (MSC) の役割を検討しました。肝細胞癌細胞ライン シートに加えて別の 2 つの細胞シートを作成: 肝細胞癌細胞、骨髄 MSCs (BMMSCs)、肝細胞と臍帯 MSCs (UCMSCs) のシートのシート。肝細胞癌細胞と MSCs の両方の組み合わせを持つシートは、担癌動物を作り出すことができます。MSCs を追加形成された腫瘍のサイズが減少し、この腫瘍の開発に悪影響を与える使用の MSCs のソースによって異なります。これは腫瘍の管理とコントロールの特定の MSC のサブタイプの細胞シートを利用できることを示します。

ラット移植細胞シートの腫瘍:
、移植後 1 ヶ月ラットの肝臓のすべての移植細胞シートは、腫瘍 (図 3) を開発しました。Hepg2 細胞 HepG2/BMMSC、UCMSC/HepG2 細胞シートから発達した腫瘍の平均のサイズは 4.5 cm、4 cm と 2.5 cm でそれぞれ10.
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Watch this Scientific Journal Video about Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo at JoVE.com

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

ここでは、細胞シート技術を用いた生体内で癌モデルを開発するためのプロトコルを提案する.このようなモデルは、抗癌剤治療の評価のため非常に役に立つかもしれない。

生体内で肝細胞癌の開発の間葉系幹細胞シートの可能性を探る

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

exploring-potential-mesenchymal-stem-cell-sheet-on-development

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

5.8K ビュー.  Ministry of National Guard Health Affairs. ここでは、細胞シート技術を用いた生体内で癌モデルを開発するためのプロトコルを提案する.このようなモデルは、抗癌剤治療の評価のため非常に役に立つかもしれない。

ビデオ: Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

肝炎症と線維症の設定に起因する肝細胞癌の腫瘍性肝細胞誘発性オルコトピックマウスモデル

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

がん研究

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the&#160;in vivo&#160;situation compared to traditional 2D cultures.&#160;Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.&#160;

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

神経癌の生物模倣モデルが、調整可能な生体特性を有する3D環境における腫瘍-ストロマ相互作用を研究する

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

免疫不全マウスの皮下および肝内ヒト肝細胞癌異種移植片の生成

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

医学

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

ザ・<em&gt;卵内</em肝細胞癌の効率的な異種移植モデルとして&gt;ニワトリ絨毛尿膜（CAM）アッセイ

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

肝細胞癌転移の「患者様」同所同系マウスモデル

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3 D ダイナミック文化システムの多層間葉系幹細胞シートの構築

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

生物工学

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

クローン性増殖のためのCD133 +肝幹細胞の単離

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

生物学

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

ラットモデルにおける部分的な術後後の漏れを減らすために脂肪組織由来幹細胞シートの移植

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

ヒト胚性幹細胞からの肝細胞様細胞誘導の効率的な方法

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...

免疫・感染学

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.