このプロジェクトの資金は、研究と網膜の研究財団のためのクレイトン財団によって提供されます。

1。網膜芽細胞腫のtumorspheresの準備<ol><li> IRB承認されたプロトコルの下で網膜芽細胞腫の腫瘍のサンプルを入手。 </li><li>新鮮定義されている幹細胞のメディアを準備する（Neurobasal -メディア、1X B - 27サプリメントマイナスビタミンA、1X非必須アミノ酸溶液、1X L -グルタミン、20 ng / mLのEGF、10 ng / mlのbFGFを）。 </li><li>機械的に滅菌、組織培養処理した6穴プレートの横断的な技法を用いてメスで腫瘍組織を分解。すぐに組織に定義された幹細胞の培地を100μLを加え、穏やかにメスを使用してみじん切りの組織を広げる。ウェルに定義された幹細胞の培地を5mLを追加。 </li><li> 37℃でインキュベートする加湿インキュベーター中、C、5％CO 2と日常点検。いくつかtumorspheresは2-4日の経過とともに増えて、ほとんどすぐに中断腫瘍組織から発生するはず。 </li><li>毎週、5分間300xgで遠心tumorspheresは、作りたての定義幹細胞の培地に再懸濁し、そしてピペット上下10倍は大きく、集中的に壊死球を破壊し、新しい、健康的な球体を形成できるようにする。 </li></ol> 2。注射のための腫瘍細胞の調製<ol><li> 15 mLコニカルチューブに静かに培養tumorspheres、ピペットの球を注入する場合。血清学的ピペットでアップダウン優しくピペット球を混乱に10-15回。 </li><li>新しいチューブに注入ごとに少なくとも50,000個の細胞を細胞は血球計算盤を用いて注入されるようにカウントし、分注し。使用可能なセルの数に基づいて、できるだけ多くの注射の準備。 </li><li> 5分間300xgで遠心分離します。注意深く5mLのPBSで上清と再懸濁しますを吸引除去する。遠心分離や吸引を繰り返し、インジェクションあたり10μLのPBSに再懸濁します。 </li></ol> 3。動物の準備<ol><li>インスリンの注射器を用いて、グラム体重あたり1μL（GBW）でげっ歯類の麻酔のカクテルの腹腔内注射（ケタミン37.6 mg / mLの、キシラジン1.92 mg / mLの、アセプロマジン0.38 mg / mL）を管理する。鎮静になるために動物のための5分間待ちます。ハートビートをチェックし、加熱されたパッドの上に動物を置く。回復されるまで常に加熱パッド上に動物を維持する。 </li><li>鎮静した後、右眼にプロパラカイン塩酸1滴を管理する。加熱されたパッドの上に動物を交換し、1分間待ちます。 </li><li>右目にフェニレフリン塩酸1滴を管理する。加熱されたパッドの上に動物を交換し、5分間待ちます。瞳孔の散大が発生していない場合、1滴を再投与し、さらに5分待って。 </li><li> &gt; 30分後に鎮静剤で発生する可能性のある運動やけいれんの兆候、動物を監視する。運動の最初の兆候で、ケタミン塩酸（PBS中10 mg / mlに希釈）の1μL/ GBWを管理し、5分間待ちます。 </li></ol> 4。腫瘍細胞の注入<ol><li>右眼を上に向けて、ガーゼで休む頭部と、その側に顕微鏡下で麻酔した動物を置き、顕微鏡で観察するときに右眼眼底（瞳がこの時に瞳孔が開いする必要がある）の赤色の反射を得るために中心。 </li><li>軽くまぶたに二本指で同時に押し下げ、手順の残りのために着実にこの位置を保持することによって右の地球を支える。 </li><li>ピアス地球儀は、毛様扁平の面積（図1）で、ちょうど硝子体腔に脈絡膜を介して角膜輪部に隣接する結膜と強膜を介して横方向に、ルアーロックシリンジに接続されている滅菌30ゲージの針を使用する。開口部から針を外します。 </li><li>アルコール綿棒でハミルトンの針を滅菌する。ハミルトンシリンジに細胞懸濁液を描画し、針が網膜の近くに硝子のレンズの後ろに顕微鏡で視覚化されるまで、開口部に針を挿入する。このポジションで着実に針を押しながらもう一人の助けを借りて、プランジャーをゆっくり押し下げます。ニードルを取り外します。必要に応じて、開口部から任意の流体を吸収するために綿棒を使用してください。 </li><li>ゆっくりとマウスのまぶたを閉じてまぶたの圧力を解放。 </li><li>それぞれの目にヒプロメロースの1滴を投与し、回復のための加熱パッドに動物を返します。 </li></ol> 5。注射したマウスと腫瘍の収穫のモニタリング<ol><li>毎日、通常2〜4週間の注射後に明らかになると、注入白色瞳孔のための目（白乳頭反射）および/または眼周囲の膨満を調べます。 </li><li>腫瘍の増殖が検出されると、制度的なガイドラインに従って動物を安楽死させる。 </li><li>目と腫瘍がまぶたで覆われている場合、眼瞼裂の直交する二切開を行い、皮膚フラップを持ち上げるためにメスを使用してください。 </li><li>実体顕微鏡を用いて可視化の下に、角膜と水晶体を除去し、角膜輪部に周方向の切開を行い、慎重に取り澄ましたを使用して他の組織から腫瘍の質量の大部分を細かく分析zers。無菌RPMI - 1640培地で腫瘍を置きます。 </li><li>ゆっくりとソケットから開いて世界を引っ張ってピンセットを使用してください。腫瘍の浸潤を可視化できるように接続された視神経を確保するために、神経が露出するまで地球を引き出し、できるだけ長く神経を切断するはさみを使用してください。組織学的検査のための定期的な処理のために10％ホルマリンに置きます。 </li></ol> 6。代表的な結果： 網膜芽細胞腫のtumorspheresはほとんどすぐに彼らは、腫瘍塊から解放として分解された組織から表示されるようになります。 2〜4日以内に、より多くのtumorspheresが形成するとサイズが増加するが開始されます。球は、通常のものと集約（図2）周囲の明確に定義された、二次膜を示す傾向がある。 動物は通常、腫瘍が大きくなるにつれて（図3c）5-8週間注射後に周囲の組織の世界と膨張の拡大が続く、4週間後の注入（図3b）内に白色瞳孔を呈する。 <img src="/files/ftp_upload/2644/2644fig1.jpg" alt="図1"/> 図1。プロトコルで参照されている機能に焦点をマウスの眼の断面図。 <img src="/files/ftp_upload/2644/2644fig2.jpg" alt="図2"/> 図2 in vitroでのヒト網膜芽細胞腫細胞の培養）は4倍で撮像、 およびb）10倍対物レンズ倍率。網膜芽細胞腫、原発腫瘍の細胞は規則的な回転楕円体の形状とさわやかな外膜とtumorspheresを生成する。スケールバーは）500μmを表し、a とb）は200μm。 <img src="/files/ftp_upload/2644/2644fig3.jpg" alt="図3"/> 図3 RAG2のアイ- / - 、B）を通常の機能を示すマウス）硝子体腔内の腫瘤を示す白色瞳孔、 およびc）大規模な腫瘍塊は、関連する眼周囲の膨満感、眼内出血で地球儀を充填。

<ol>
	<li>Ch&#232;vez-Barrios, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+and+Nonmetastatic+Models+of+Retinoblastoma.">Metastatic and Nonmetastatic Models of Retinoblastoma.</a> Am. J. Pathol. 157, 1405-1412 (2000).</li><li>Suber, M. L., Hurwitz, M. Y., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+consequences+of+intraocular+administration+of+modified+adenoviral+vectors.">Immune consequences of intraocular administration of modified adenoviral vectors.</a> Hum. Gene Ther. 12, 833-838 (2001).</li><li>Mallam, J. N., Hurwitz, M. Y., Mahoney, T., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Gene+Transfer+into+Retinal+Cells+Using+Adenoviral+Vectors:+Dependence+on+Receptor+Expression.">Efficient Gene Transfer into Retinal Cells Using Adenoviral Vectors: Dependence on Receptor Expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1680-1687 (2004).</li></ol>

動物実験は医学IACUC委員会のベイラー大学で定められたガイドラインおよび規制に準拠して行った。

ここに記載された技術は、彼らの眼内、硝子体内環境での伝搬の網膜芽細胞腫の腫瘍を促進する。眼内注射の技術は、網膜芽細胞腫由来細胞株1と同様に眼遺伝子治療2,3のためのウイルスベクターを提供するから腫瘍を作成するために、過去に使用されています。この手法は、これで正常に原発腫瘍と注射tumorspheresのだけでなく、異種移植片腫瘍のシリアル伝播から細胞を直接注入することにより、人間の網膜芽細胞腫の腫瘍を生成するために利用されている。腫瘍形成（通常は白色瞳孔）の目に見える証拠は、通常、最初に軌道を取り巻く世界および/または組織の眼内出血や膨満5〜8週間以内に開発した後、4週間以内に記載されています。注射されたマウスの少数派は、まぶたの永久閉鎖につながる、充血した目を傷つける。これらのケースでは、膨満は、腫瘍形成の唯一の徴候である。 確立された異種移植腫瘍の増殖が非常に成功した場合もマウス異種移植腫瘍の確立は、すべての人間の網膜芽細胞腫の腫瘍を持つ成功していない。この観察は、そのような差別やその他の未知の要因の侵襲性やレベルなどの原発腫瘍の特定の特性が、、マウス眼球の環境中に残留するためにこれらの腫瘍の能力に影響を与える可能性がありますことを示唆している。 人間の網膜芽細胞腫の腫瘍から取得することができる組織の量はかなり小さいです、そしてそのような限られた寿命、固形腫瘍の組織像の消失と細胞の表現型の変化として、 体外培養ヒト網膜芽細胞腫の細胞への能力に大きな制限があります。このプロトコルは、ヒトの腫瘍を伝播し、ヒト疾患のマウスモデルを確立するために比較的単純な方法を提供します。これは、in vitroおよび網膜芽細胞腫と患者の腫瘍外の長いメンテナンスの生物の in vivo実験におけるさらなるできます。

<table border="1"><tbody><tr><th>の名前</th><th>会社</th><th>カタログ＃ </th><th>コメント</th></tr><tr><td>塩酸2.5％フェニレフリン</td><td>ボシュロム</td><td> 053から11 </td><td>点眼剤</td></tr><tr><td> 30 - GAニードル</td><td> BD </td><td> 305128 </td><td>レギュラーベベル</td></tr><tr><td> 10 mLのルアーロックシリンジ</td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 3/10-ccインスリン注射器</td><td> BD </td><td> 328431 </td><td></td></tr><tr><td>アルコール綿棒</td><td> BD </td><td> 326895 </td><td></td></tr><tr><td> 6ウェルプレート、組織培養用</td><td> BD </td><td> 353046 </td><td></td></tr><tr><td>プロパラカイン塩酸0.5パーセント</td><td>バトラーAHS </td><td> 017239 </td><td>点眼剤</td></tr><tr><td>ケタミン塩酸100mg/mL </td><td>フォートドッジ</td><td> 4402A </td><td></td></tr><tr><td> 10μLハミルトンシリンジ</td><td>ハミルトン社</td><td> 7648〜01 </td><td></td></tr><tr><td> 32 - GAハミルトンニードル</td><td>ハミルトン社</td><td> 7803〜04 </td><td>カスタム長さ - 0.5&quot; </td></tr><tr><td> Neurobasalメディア</td><td>インビトロジェン</td><td> 10888-022 </td><td></td></tr><tr><td> B - 27サプリメントマイナスビタミンA、50X </td><td>インビトロジェン</td><td> 12587-010 </td><td></td></tr><tr><td> RPMI - 1640メディア</td><td> Mediatech </td><td> 10から040 - CV </td><td></td></tr><tr><td>非必須アミノ酸溶液、100X </td><td> Mediatech </td><td> 25から025 - CI </td><td></td></tr><tr><td> 100X、L -グルタミン、 </td><td> Mediatech </td><td> 25から005 - CI </td><td></td></tr><tr><td>使い捨て＃11メス</td><td> Miltex </td><td> 4から411 </td><td></td></tr><tr><td>げっ歯類の麻酔のコンボ</td><td> N / A </td><td> N / A </td><td>薬局製剤（ケタミン37.6 mg / mLの、キシラジン1.92 mg / mLの、アセプロマジン0.38 mg / mL）は、社内における</td></tr><tr><td>組換えヒト上皮成長因子（EGF） </td><td> STEMCELLテクノロジーズ</td><td> 02633 </td><td> 10μg/ mLの原液で再構成する</td></tr><tr><td>組換えヒト塩基性線維芽細胞増殖因子（bFGF） </td><td> STEMCELLテクノロジーズ</td><td> 02634 </td><td> 10μg/ mLの原液で再構成する</td></tr><tr><td> OMS - 75手術用顕微鏡</td><td>トプコンメディカルシステムズ</td><td> OMS - 75 </td><td>このモデルは廃止されています</td></tr><tr><td> 10％ホルマリン</td><td> VWR </td><td> 95042-908 </td><td></td></tr></tbody></table>

establishment and propagation of human retinoblastoma tumors in immune deficient mice

定義された幹細胞培地で培養網膜芽細胞腫の腫瘍細胞は、限られた時間のために栽培し、維持することができる主tumorspheresを生じさせる。元の腫瘍と比較すると、これらの培養tumorspheresは著しく異なる細胞の表現型を示すことができる。培養細胞は、新しい腫瘍を形成する能力を持っているデモは、その培養細胞モデルを元の腫瘍の生物学を確保することが重要です。 / - -免疫欠損マウスここではRAG2を用いた in vivo でのヒト網膜芽細胞腫の腫瘍を伝播するためのプロトコルを提示する。低継代またはマウスの眼の硝子体腔内に直接注入されたばかりの収穫、人間の網膜芽細胞腫の腫瘍から得られた細胞の培養ヒト網膜芽細胞腫のtumorspheresは2-4週間以内に腫瘍を形成する。これらの腫瘍を採取し、どちらさらにin vivoでのマウスの目に継代またはin vitroで tumorspheresとして成長させることができる。伝播が正常に少なくとも三つの通路ため、さらに実験のための人間の網膜芽細胞腫の組織の継続的なソースを確立するために行われている。 ウェズリーS.ボンドとLalita Wadhwaは、共同最初の著者です。

Watch this Scientific Journal Video about Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice at JoVE.com

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

メソッドは、マウスにヒトの網膜芽細胞腫の腫瘍を伝播するために記述されています。腫瘍細胞は、直接免疫欠損マウスの眼に注入されています。二次腫瘍が正常に直接ヒト腫瘍および培養tumorspheresから採取した細胞の両方を使用して確立されている。

免疫不全マウスにおけるヒト網膜芽細胞腫の腫瘍の確立と伝播

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

establishment-propagation-human-retinoblastoma-tumors-immune

Research

JoVE Journal

Medicine

11.7K ビュー.  Baylor College of Medicine. メソッドは、マウスにヒトの網膜芽細胞腫の腫瘍を伝播するために記述されています。腫瘍細胞は、直接免疫欠損マウスの眼に注入されています。二次腫瘍が正常に直接ヒト腫瘍および培養tumorspheresから採取した細胞の両方を使用して確立されている。

ビデオ: Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

検証済みの内視鏡スコアリングシステムを使用して、大腸炎および結腸直腸腫瘍の重症度を評価するためにマウスでの柔軟な大腸内視鏡検査

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

医学

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

免疫無傷のヒトMUC1トランスジェニックマウスにおける侵襲移行細胞膀胱癌の誘導：免疫療法の開発のためのモデル

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

蛍光SAPC-DOPSナノベシクルを用いて脳腫瘍、関節炎のin vivo光イメージング中

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

生きたマウスにおける大腿骨髄吸引

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

解剖学的に正確な作成と人間の脳の腫瘍の再現性頭蓋内異種移植片

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

全組織生検からヒト上皮EnteroidsとColonoidsの確立

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

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 ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

フローサイトメトリーを用いたひと脂肪組織での免疫細胞のキャラクタリゼーション

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.