SW1353軟骨肉腫細胞株由来の細胞を親切に教授PCW Hogendoornとライデン大学、オランダの教授J.Bovéeによって提供された。私たちは、プロトコルの概要の専門描画にJ. MestachとG. Wagemans優れた技術支援のために、そしてG.デBruyneに感謝します。

概要については、図1を参照してください腫瘍材料 1。腫瘍サンプルの取得と準備患者材料の使用については、倫理委員会の承認が必要であり、インフォームドコンセントは、患者から得られなければならない。 <ol><li>介入時の収穫代表材（最小1センチメートル3）、生検または肉腫の切除のどちらか。生検の適切な部位は、ダイナミックコントラストMRIを使用して定義されます。 </li><li> 1,000 Uペニシリン及び1 mg / mlのストレプトマイシンを補充したDMEMを含む滅菌バイアルにすぐに材料を置きます。 </li><li>ラボに - 直ちにバイアルを輸送（90分30）。 </li></ol>すべての次の手順は、層流の下で実行されます。 <ol start="4"><li>無菌のペトリ皿にバイアルの内容物を注ぐ。 </li><li>最大1メートルの小さな部分に腫瘍サンプルを入れて滅菌メスを用いてm 3である。彼らは組織の更なる処理を損なう傾向があるように、すべての石灰化部分を削除。 </li><li>ランダムCAM上のアプリケーションのための10の腫瘍移植片を取る。 </li></ol> 2。腫瘍由来の単一細胞懸濁液の調製おおよその時間：3時間<ol><li>サンプルの残りの組織を量る。 </li><li>入り、2 - 解離チューブ内の組織を4g、複数のチューブが残りのすべての腫瘍組織を消化するために必要とされてもよい。 </li><li> 2の消化のために - 組織の4グラム、2.5ミリリットルコラゲナーゼ2溶液（500 U / mlのRPMI 1640）と2.5ミリリットルのDNase溶液（22 KU / mlのRPMI 1640）を追加します。 </li><li>解離h_tumorプロトコルに従って組織を処理します。管を30分間穏やかに振盪しながら37℃でCO 2インキュベーター内℃でのインキュベーションの2サイクルを含む。 </li><li> 150μmのセルストレーナーを通して、残りの懸濁物をろ過する。 </li><li> 5マイル、1,000 rpmでライセートを遠心N。 </li><li>上清を取り除きます。 </li><li>正規懸濁液とのインキュベーションの10分を可能にする、赤血球溶解バッファー（ELB）10mlにペレットを再懸濁する。 </li></ol>注：赤血球溶解緩衝液は、以下のように製造される：0.037グラムのEDTA、0.99グラムK 2 HPO 4、千ミリリットル水に8.29グラムの NH 4 Clをを追加し、0.22μmの減圧フィルタ、7.3にpHを調整するために10 N水酸化ナトリウムを使用フィルタ。 4℃で保存<ol start="9"><li>反応を停止さ培地（DMEM、10％ウシ胎児血清、100 U / mlのペニシリン、および100μg/ml/ mlのストレプトマイシンを補充した）25 mlを加える。 </li><li> 5分間1,000 rpmでサスペンションを遠心分離。ペレットの色は今白でなければなりません。 </li><li>上清を取り除きます。 </li><li>ペレットに培地5mlを追加します。 70μmのセルストレーナーを通してサスペンションをフィルタリング。 </li><li>自動セルカウンターを用いて生細胞/ mlの数を数える。 </li></ol> 3。 C言語アンサー細胞株 SAOS2骨肉腫細胞（​​ATCC番号：HTB-85）強化緑色蛍光タンパク質を発現し、製造業者のプロトコルに従って細胞株ヌクレオキットVを用いたpEGFP-C1ベクターによってエレクトロポレーションした。安定な細胞株を確立するために、トランスフェクトされた細胞は、以前に確立プロトコル20に沿って、4週間のG418（1 mg / ml）で選択した。加えて、ソートは、製造業者の指示に従って、蛍光活性化細胞選別（FACS）によって行った。 SW1353軟骨肉腫細胞株（ATCC番号：HTB-94）からの細胞または作りたて腫瘍単一細胞懸濁液は、赤色蛍光親油性膜染色を用いて標識されています。 <ol><li>溶液;トリプシン（0.5％重量/体積）エチレンジアミン四酢酸（0.2％重量/容量EDTA）を使用して古典的な方法で培養フラスコから細胞を取り出します。 </li><li> 1,000 rpmで5分間、細胞懸濁液を遠心分離する。 </li><li> supernを削除atant。 </li><li>無血清培地1mlと5μlのDiIを（Vybrant赤）/ 10 6個の細胞を追加します。 </li><li>アルミホイルでバイアルを包み、37℃で10分のためにCO 2インキュベーターに入れて</li><li> 1,000 rpmで5分間、再び遠心し、上清を除去します。 </li></ol>漿尿膜アッセイ 1。卵のインキュベーション<ol><li>卵の95％の受精を保証する地元商業孵化場から受精卵が必要となります。 </li><li>受精卵は10日まで室温で、最大保存することができます。 </li></ol>注：インキュベーションを開始する前に、汚れ、羽や排泄物を慎重にむしろ柔らかい表面構造よりもラフを持って紙タオルと乾拭きで機械的に卵の殻から削除されます。 70％変性エタノールまたは他の洗浄試薬で卵をふくと、大幅にニワトリ胚の生存率を低減します。 <ol start="3"><li> incubに卵を入れて片側にator、上面のマーキング。卵はインキュベーターに入れられた日は、胚発生の0日に指定されます。 </li><li> 37.8℃のインキュベーション温度、および47％と湿度を設定する。自動回転オプションがオフになっていることを確認してください。 </li></ol> 2。卵のオープニング長さ：25卵は±60分開発3日目、卵は層流の下で開かれます。膜は殻に固執する傾向があるように、CAMへのダメージがさらに発達段階の結果で卵を開く。赤外線ランプは、手順の間に卵を暖かく保つために使用されます。無菌性を改善するために、我々は手の手袋の使用をお勧めします。 <ol><li>上向きに顕著な側面と、ゆで卵を置きます。 </li><li>卵の先端に挿入された18 G針を卵白の2ミリリットルを除去することにより、CAMのレベルを下げます。針は、複数の穿孔後鈍である場合卵の殻には、針を交換してください。そうでない場合は、あまりにも力が卵黄またはシェルの破壊への損傷、その結果、発揮されなければならない。時には、針はchalazae、卵白に卵黄を中断2スパイラルバンドの一つによってブロックされています。これは、ブロックが解決されるまで穏やかに戻ってプランジャを押し下げることによって解決することができる。卵黄をシリンジ内に吸引されている場合は、注射針と同様に注射器を交換してください。時には、胚は、このイベントの後に死ぬ。卵白の不十分な量が除去されると、シェルが除去されると、CAMが破損します。 </li><li>ウィンドウを削減しながらCAM上にシェル粒子がこぼれるのを防ぐためにシェルのマークされた上側の半透性接着フィルムを適用する。 </li><li>卵の表面に小さな千枚通しと導入孔を作る。 </li><li>無菌先鋭外科はさみのペアを使用して、シェルが1cm 2の窓をカットします。 </li><li>胚の脈動心臓や隣接血管が目で観察することができます卵黄の電子表面。非受精卵または死んだ胚を削除します。血管の中断された態様は、死んだ胚を特徴付ける。 </li><li>半透性接着フィルムでウィンドウを封印。シェルは、針の挿入中に割れていない場合、それは、穿刺部位をカバーする必要はない。 </li></ol> 3。接種手順時間：細胞株当たり±1時間ニワトリ胚発生の9日目、卵は層流の下に接種する。 CAMに広がる癌細胞の評価は、接種時に限られた表面への細胞の封じ込めを必要とします。マトリゲルは、しばしば実験的な細胞培養条件下で使用されるEngelbreth-ホルム - 群れマウス肉腫から細胞外マトリックス（ECM）ゲルである。それが冷却する流体であるが、20に運ばとき熱的に活性化して重合を受ける - 40°C、従って安定なゲルを形成する。間に実験は、マトリゲルは、氷の融解を含むボックスに保持される。 注：私たちは、穴あきカバーガラスを使用しないよう主張している。我々は、膜自体の細胞のプラスチックディスクこうしてバイアス成長性にグラフト癌細胞の付着と成長を観察した。 <ol><li> 10 6 cells/100μlのECMゲルの濃度で冷却ECMゲルで細胞ペレットを再懸濁します。溶ける氷の上でこのソリューションを保つ。 </li><li>層流の下で無菌手術一対の半透膜の切除部分。接着フィルムが完全に除去されている場合、これは汚染による胚の死の割合が大きいことになる。 </li><li>上皮細胞層を除去するために、滅菌したガラス棒で軽くシェルウィンドウ下CAM右をタッチします。滅菌したガラス棒でCAMに触れると、より扱いやすさと経験を必要とするメスN°11を使用して、より少ない外傷であることが分かる。小さな出血が発生することがあります。 </li><li> A腫瘍物質dding。 </li><li>腫瘍移植の場合：滅菌ピンセットのペアでCAMへの組織の一つでそっと下片、それを圧迫せずに。 </li><li> ECMゲル手順について：ECMゲルは、アプリケーション間で重合することができ、互いの上に、CAMに細胞-ECMゲル懸濁液を4倍25μLを加える。このようにして癌細胞を含有する接着性プラークが作成される。 </li><li>半透接着フィルムでもう一度シェルウィンドウをシールします。 </li></ol> 4。 CAMのイメージングと収穫長さ：25卵のために2時間ニワトリ胚発生の16日目で、カムが収穫されます。層流の下で作業する必要はありません。 <ol><li>手術用はさみでウィンドウを拡大する。負傷した血管の出血で、その結果、そうでなければCAMが損傷されますが、低すぎると切らないようにしてください。 </li><li> 300を追加- PBS Dの500μlのCAMの宗派へのピペットで接種材料を含むイオンである。まだ蛍光プローブを使用しないときは、この膜が硬くなるように、緩衝ホルムアルデヒドは、膜の切断を容易に使用することができる。 </li><li> GFPまたはDSRフィルタ、フィルタなしの両方を使用してデジタルカラーカメラを搭載したステレオ蛍光顕微鏡を通して卵におけるCAMの写真を、作る。腫瘍移植の場合は、すべての写真はフィルターなしで作られています。 LED照明によって、上記からの照明が強く（ 図2）撮影時をお勧めします。 </li><li>標識された細胞のレコード腫瘍細胞遊走。腫瘍の境界が不規則で擦り切れたかもしれません。散乱した細胞は、腫瘍の境界付近に存在してもよい。いくつかの標本では、腫瘍細胞の行（ 図3）を観察することができる。 </li><li>シェルに対して嘘国境で滅菌はさみのペアを持つ膜をカットします。 </li><li> PBS Dで埋めたりのためにバッファリング滅菌ペトリ皿で収穫されたCAMを置きmaldehyde。 </li><li>上側とし、フィルタなしの両方のCAMの下側、両方の写真を作成します。 </li><li>少なくとも72時間のためにバッファリングされたホルムアルデヒドとラベルされた容器内に膜を置く。 </li><li>パラフィンでカム埋め込み、組織学的検査のためにそれらを準備します。切断面は、カセットの底に平行であることを確認し、結節の中心に腫瘍移植片をカットするように注意してください。そうでない場合は、CAMと腫瘍移植片の間の相互作用は表示されません。同戦略は、ECMゲルプラークに適用されます。切刃との対向面にはプラークに垂直でなければなりません。 </li></ol> 5。組織学的評価必要に応じて、ヘマトキシリン​​ - エオシン染色およびKi-67免疫組織化学は、さらに明確化のために行った。免疫組織化学係る自動化されたスライド染色を用いて、厚さ3.5μmでホルマリン固定パラフィン包埋組織切片上で実施した製造元の指示である。のKi-67（クローンMIB-1、希釈1/100）にマウスモノクローナル一次抗体を用いた。熱誘導エピトープ検索は、Cellコンディショニング1を用いて行った、可視化は、製造業者の指示に従って、ユニバーサルDAB検出キットで達成された。組織切片のDehydratationは、自動化されたカバーガラスを用いて行った。 SAOS2とSW1353細胞株、Ki67の陽性数とKi67の陰性細胞nuclei/100μmの2は 3つのゾーンで計数した場合：1近いCAMの国境に、一面との中央に1を閉じるECMゲルプラーク。この手順は、それぞれのKi-67染色したスライドの6倍速繰り返した。増殖の指標をカウントした細胞の総数であるKi67陽性細胞の数を割っスライドごとに決定した。 壊死はファディ伴う細胞核内クロマチンの微細分散の損失によって定義され個別のセルの余白のNG。異種移植の場合、壊死が、完全な（しばしば表面で）部分的、または欠席として記録されています。 CAMへの腫瘍細胞の浸潤は、CAMの中胚葉内の腫瘍細胞の観察により定義され、有無であると採点される。 三項目は、ホストの動作のために採点されます。線維芽細胞の浸潤は、細長い細胞がCAMを残して、腫瘍移植片/プラークを侵略として定義され、有無であると採点される。血管の内部成長は核ニワトリ赤血球の存在によって特徴付けひよこ血管を、増殖の内部成長として観察することができます。

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P., Sidebottom, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+invasion+and+intramesenchymal+infiltration+of+the+chick+embryo+chorioallantois+by+tumor+cell+lines.">Transepithelial invasion and intramesenchymal infiltration of the chick embryo chorioallantois by tumor cell lines.</a> Cancer Research. 42, 1826-1837 (1982).</li><li>Deryugina, E., Quigly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+model+systems+to+study+and+visualize+human+tumor+cell+metastasis.">Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis.</a> Histochemistry and Cell Biology. 130, 1119-1130 (2008).</li><li>Knighton, D., Ausprunk, D., Tapper, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avascular+and+vascular+phases+of+tumor+growth+in+the+chick+embryo.">Avascular and vascular phases of tumor growth in the chick embryo.</a> British Journal of Cancer. 35, 347-356 (1977).</li><li>Dohle, D. S., Pasa, S. D., Gustmann, S., Laub, M., Wissler, J. H., Jennissen, H. P., D&#252;nker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+ex+ovo+culture+and+ex+ovo+CAM+assay:+how+it+really+works.">Chick ex ovo culture and ex ovo CAM assay: how it really works.</a> J. Vis. Exp. (33), e1620 (2009).</li><li>Balke, M., Neumann, A., 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=Morphologic+characterization+of+osteosarcoma+growth+on+the+chick+chorioallantoic+membrane.">Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane.</a> BMC Research Notes. 3, 58 (2010).</li><li>Hendrix, A., Maynard, D., 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=Effect+of+the+secretory+small+GTPase+Rab27B+on+breast+cancer+growth,+invasion,+and+metastasis.">Effect of the secretory small GTPase Rab27B on breast cancer growth, invasion, and metastasis.</a> Journal of the National Cancer Institute. 102, 866-880 (2010).</li><li>Ausprunk, D., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+of+normal+and+neoplastic+tissues+grafted+to+the+chick+chorioallantois:+role+of+host+and+preexisting+graft+vessels.">Vascularization of normal and neoplastic tissues grafted to the chick chorioallantois: role of host and preexisting graft vessels.</a> American Journal of Pathology. 79, 597-618 (1975).</li><li>Lokman, N. A., Elder, A. S. F., Riciardelli, C., Oehler, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+Chorioallantoic+membrane+(CAM)+Assay+as+an+in+vivo+model+to+study+the+effect+of+newly+identified+molecules+on+ovarian+cancer+invasion+and+metastasis.">Chick Chorioallantoic membrane (CAM) Assay as an in vivo model to study the effect of newly identified molecules on ovarian cancer invasion and metastasis.</a> International Journal of Molecular Sciences. 13, 9959-9970 (2012).</li><li>Vargas, A., Zeisser-Labou&#232;be, M., Lange, N., Gurny, R., Delie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick+embryo+and+its+chorioallantoic+membrane+(CAM)+for+the+in+vivo+evaluation+of+drug+delivery+systems.">The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems.</a> Advanced Drug Delivery Reviews. 59, 1162-1176 (2007).</li><li>Hagedorn, M., Javerzat, S., 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=Accessing+key+steps+of+human+tumor+progression+in+vivo+by+using+an+avian+embryo+model.">Accessing key steps of human tumor progression in vivo by using an avian embryo model.</a> Proceedings of the National Academy of Sciences U.S.A. 102, 1643-1648 (2005).</li><li>De Wever, O., Hendrix, A., 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=Modeling+and+quantification+of+cancer+cell+invasion+through+collagen+type+I+matrices.">Modeling and quantification of cancer cell invasion through collagen type I matrices.</a> International Journal of Developmental Biology. 54, 887-896 (2010).</li><li>Albini, A., Benelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemoinvasion+assay:+A+method+to+assess+tumor+and+endothelial+cell+invasion+and+its+modulation.">The chemoinvasion assay: A method to assess tumor and endothelial cell invasion and its modulation.</a> Nature Protocols. 2, 504-511 (2007).</li><li>Hanahan, D., Coussens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessories+to+the+crime:+functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 21, 309-322 (2012).</li></ol>

著者らは、開示することは何もありません。

接種と収穫時接種の日はECMゲル（36カム）でSAOS2を使用して実行し、胚発生の5日目と10の間で変化させたタイミング。 インキュベーション9日の前に、CAMは一貫して私たちが適用されたECMゲルをサポートするのに十分な大きさではなかった。収穫では、腫瘍細胞は時々深いCAMから取得しなければならなかった、といくつかのECMゲルサンプルは、卵白やCAM?...

肉腫は、治療抵抗1,2による高い死亡率と結合組織のまれな腫瘍である。患者の生存率の進歩は、その低い年間発生率、彼らの幅広い多様性、および肉腫細胞をin vitroで 3,4 での培養には難しいことがあると報告されているという事実によって妨げられている。 前臨床治療の評価のための培養細胞の使用は、 インビトロで新しい、明らかに活性な分子が常に臨床に結果を反映していないことを明らかにした。さらに、遺伝子発現アレイによって明らかにされたゲノム収差は常に5-7個々の患者の腫瘍の行動特性に相関はありません。これらの問題を試してみて、解決するために、パーソナライズされた薬は異種移植モデル8-12のために増加し、検索に反映されている重要で得ています。 インビボアッセイにおけるcの間の複雑な相互作用を反映するという利点を有する癌の増殖と浸潤13のために必要なアンサー細胞および固形腫瘍における宿主組織環境。現在、我々は肉腫14,15のため再現性の異種移植モデルとしてChorioの-尿膜アッセイ（CAMアッセイ）の使用を検討する。このアッセイは、広く腫瘍の血管新生16,17の研究のために使用される。他の研究では、異なるプロトコル18,19に従って成長や血管新生の著しい違いを観察しながら、文献で ​​は、しかし、我々は、このアッセイのために、異なるプロトコルを発見した。 本稿では、腫瘍移植片、腫瘍由来の単一細胞懸濁液と確立された肉腫細胞培養物を用いた細胞の挙動にCAMアッセイの条件を変化させる効果を調べる。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Cell Line Nucleofector Kit V</td>
			<td>Amaxa</td>
			<td>VCA-1003</td>
		</tr>
		<tr>
			<td>collagenase 2 solution (500 U/ml RPMI 1640)</td>
			<td>Sigma Aldrich</td>
			<td>C6885</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41965-039</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
		</tr>
		<tr>
			<td>Dnase solution</td>
			<td>Sigma Aldrich</td>
			<td>DN25</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>11811031</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E1270</td>
		</tr>
		<tr>
			<td>mouse primary monoclonal antibody Ki67</td>
			<td>Dako Denmark</td>
			<td>MIB-1</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fluka</td>
			<td>D76240</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>20012019</td>
		</tr>
		<tr>
			<td>PBSD</td>
			<td>Invitrogen</td>
			<td>14040083</td>
		</tr>
		<tr>
			<td>peGFP-C1 vector</td>
			<td>Clontech</td>
			<td>632470</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15140163</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Invitrogen</td>
			<td>22409-015</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Invitrogen</td>
			<td>25300054</td>
		</tr>
		<tr>
			<td>Vybrant cell-labeling DiI</td>
			<td>Lifetechnologies</td>
			<td>22885</td>
		</tr>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
		</tr>
		<tr>
			<td>digital color camera</td>
			<td>Leica</td>
			<td>DFC 340 FX</td>
		</tr>
		<tr>
			<td>Digital Egg Incubator</td>
			<td>Auto Elex Co</td>
			<td>R-COM 50</td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>BD Biosciences</td>
			<td>FACSAriaIII</td>
		</tr>
		<tr>
			<td>Gentlemacs C-Tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator User Manual containing h_tumor protocol</td>
			<td>Miltenyi Biotech</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>semipermeable adhesive film (Suprasorb F)</td>
			<td>Lohmann&amp;Rauscher</td>
			<td>20468</td>
		</tr>
		<tr>
			<td>stereo fluorescence microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
		</tr>
		<tr>
			<td>Tissue-Tek Film automated Coverslipper</td>
			<td>Sakura</td>
			<td>6400</td>
		</tr>
		<tr>
			<td>ultraView Universal DAB Detection Kit</td>
			<td>Ventana Medical Systems Inc</td>
			<td>760-500</td>
		</tr>
		<tr>
			<td>Ventana Automated Slide Stainer</td>
			<td>Ventana Medical Systems</td>
			<td>Benchmark XT</td>
		</tr>
	</tbody>
</table>

the in ovo cam-assay as a xenograft model for sarcoma

肉腫は、すべての新しい治療法の開発を妨げ、自然の中で異質である非常にまれな疾患である。肉腫患者がこの病気のために再現性と低コストの異種移植モデルの開発で現在の関心を説明、成層後のオーダーメイド医療のための理想的な候補です。ニワトリ漿尿膜は、種特異的な制限なしにグラフトされた組織や細胞を維持することのできる自然な免疫不全宿主である。また、容易にアクセス、操作及び光と蛍光実体顕微鏡を用いて撮像される。組織学はさらに異型細胞間相互作用の詳細な分析を可能にします。 このプロトコルは、新鮮な肉腫由来の腫瘍組織、それらの単一細胞懸濁液と、恒久的かつ一時的な蛍光標識確立肉腫細胞株（SAOS-2、SW1353）と絨毛尿膜のグラフト卵で詳細に説明しています。ひよこ生存ラットESは75％アップしている。モデルは、移植片（生存能力、Ki67の増殖指数、壊死、浸潤）とホスト（線維芽細胞の浸潤、血管内殖）挙動を研究するために使用されます。単一細胞懸濁液の移植ローカライズについては、ECMゲルは不活性封じ込め材に比べて大きな利点を提供します。 Ki67の増殖指数は、CAMの表面からの細胞の距離とCAM、治療製品の添加の時間枠を決定し、後者上のアプリケーションの継続期間に関連する。

CAMの評価腫瘍移植片はCAM（ 図2A）に付着なる。患者材料からの単一細胞懸濁液は、頻繁に干し、やや隆起したプラーク（ 図2D）が表示されます。 CAMの切除後、膜のしわ（2Eと2Fの図 ）が発生をマーク。 商業細胞株についてプラークは細胞増殖を示す時間より不透明になる。 ECMゲル中の異なる細胞株は...

Watch this Scientific Journal Video about The In ovo CAM-assay as a Xenograft Model for Sarcoma at JoVE.com

The In ovo CAM-assay as a Xenograft Model for Sarcoma

ザ<em&gt;卵で</em&gt;絨毛尿膜（CAM）は、新たな肉腫由来の腫瘍組織、それらの単一細胞懸濁液、および恒久および一時蛍光標識された確立された肉腫細胞株でグラフトされている。モデルは、移植片（生存能力、Ki67の増殖指数、壊死、浸潤）とホスト（線維芽細胞の浸潤、血管内殖）挙動を研究するために使用されます。

ザ<em&gt;卵で</em肉腫のためXenograftmodelとして&gt; CAMアッセイ

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

the-in-ovo-cam-assay-as-a-xenograft-model-for-sarcoma

Research

JoVE Journal

Medicine

The In ovo CAM-assay as a Xenograft Model for Sarcoma

25.3K ビュー.  Ghent University Hospital. ザ卵で絨毛尿膜（CAM）は、新たな肉腫由来の腫瘍組織、それらの単一細胞懸濁液、および恒久および一時蛍光標識された確立された肉腫細胞株でグラフトされている。モデルは、移植片（生存能力、Ki67の増殖指数、壊死、浸潤）とホスト（線維芽細胞の浸潤、血管内殖）挙動を研究するために使用されます。

ビデオ: The In ovo CAM-assay as a Xenograft Model for Sarcoma

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

そのような自閉症や統合失調症のような一般的な疾患でのde novo変異を同定するための戦略

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

医学

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

マウスにおける頭蓋内異種移植研究のための簡単​​なガイドスクリュー方式

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

単離された灌流脊椎動物の網膜のためのストレスモデルとしてグルタミン酸および低酸素症

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

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 ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

腫瘍の増殖、浸潤及び血管新生を研究するために、ニワトリでのヒト多発性骨髄腫異種移植モデルの確立

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

急性呼吸窮迫症候群のためのモデルとして豚のオレイン酸注入

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Methods Article

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