我々は、臨床検体を得るための彼女の貴重な援助のためにローナレンショウ（エジンバラの乳ユニットを）感謝。 腫瘍の材料の使用量は、ロージアンの研究倫理委員会（08/S1101/41）によって承認され、実験的がん医療センタープログラム（エジンバラ）の後援の下に得られた。 この作品は、スコットランドの財政審議会でサポートされています。 ADLは、ミスマーガレットバタードリーキー信託臨床の仲間です。

1。ラットの尾からコラーゲンを抽出<ol><li> 70％エタノールで冷凍新鮮なラットの尾を置きます。 </li><li>メス公開する腱を覆う皮膚をDeglove。 </li><li>滅菌に離れて70％エタノールで尾と場所から腱を剥ぎ取ります。 </li><li>収集した腱と酢酸（250ミリリットル0.5M酢酸に1gの腱）への転送を量る。 4℃で48hrのためにミックス℃に</li><li> 30分間10,000 gで腱/酢酸の混合を遠心し、ペレットを捨てる。 </li><li>上清に10％の等量（w / v）のNaClを追加し、ミックスは4℃で一晩放置する℃に</li><li> G. 10,000さらに30分のためのコラーゲンが豊富な、不溶性の底層とcentrifugateを収集する</li><li> 4℃で0.25M酢酸でコラーゲン豊富な材料° Cを再懸濁し、毎日二度の透析バッファーを変更すること、3日間4℃で1:1000酢酸に対してdialyse。 </li><li>さらに4℃で遠心分離（2時間、20,000 g）とストアによってコラーゲン溶液を滅菌℃に</li><li> 1mg/mlのストック濃度に滅菌1:1000酢酸の添加により、必要に応じてコラーゲンを希釈する。 </li></ol> 2。 3Dアッセイの作成 3Dアッセイは、以前は次の2発行された、細胞株のアッセイに基づいています。 <ol><li>複数のコア生検は侵襲性乳癌に対する根治的切除術の時に患者の同意から採取されています。 </li><li>目によって、1ミリメートル3フラグメントにメスを使用してコアを割る。巨視的に異なる脂肪をトリミングして捨てる。 </li><li> MEGM完全なメディアの断片を浸す。組織は、最大1時間までは、この状態で保存することができます。 </li><li>氷1mg/mlのラット尾コラーゲンでコラーゲンゲル、ミックスを作成するには、1:1000フィルタリング酢酸、3:5:1:1の比率で10倍DMEM/F12と0.22MのNaOH、0.3の最終コラーゲン濃度を作成するmg / mlの。 </li><li> 37℃インキュベーターに24ウェルプレートおよび場所の個々のウェルにコラーゲンの混合物の1.2ミリリットルを注入℃で10分間ゲル化を開始する。 </li><li> 10分後、インキュベーターから24ウェルプレートを取り外します。ゲル化コラーゲンへの腫瘍片を置き、静かに10μlをピペットチップを用いてゲルの中心にそれらを押してください。 1時間のためのインキュベーターに24ウェルプレートを返します。 </li><li> ER +腫瘍の場合は、3.2x10 -10 Mの濃度にβ-エストラジオールとMEGM完全なメディアを補完するゲルアッセイは、1時間のためにゲル化した後、慎重に各ウェルに添加培地の1.2ミリリットル紹介します。アッセイ内の最後のβ-エストラジオール濃度は、乳房組織3で見られる生理的なエストロゲンのレベルを再現するために1.6 × 10 -10 Mにequilibriateなります。 </li><li>各ゲルは、メディア内で自由にフロートすることができますので、円周方向にそれぞれのゲルを放出、各ウェルの内側のエッジの周り10μlをピペットの先端を実行します。インキュベーターに24ウェルプレートを返します。 </li><li> Exchangeは実験終了まで、メディアは週2回の補足。メディアを削除し、ホルマリン固定液を追加して実験を終了します。 </li></ol> 3。光学投影断層撮影の準備のためにアッセイの抗体染色我々は、全サンプルの染色の4の以前に発行された方式を採用している。 <ol><li> PBSで30分のためのホルマリン固定標本を洗浄してください。 33％、66％、各ステップで≥15分のための100％：0.05％ツイーン20（0.05％PBST）を含むPBSで希釈したメタノールで段階的にゲルを脱水する。 </li><li> DMSO：30％H 2自己蛍光をクエンチするために24時間、室温で2時01分03秒の比率（15％v / vのH 2 O 2）で、O 2、新しく調製MeOH中で組織をインキュベートする。 </li><li>メタノールで30分間二回のサンプルを洗浄する。 -80 ° C組織の深い部分でその抗原性を確保するために室温（RT）に戻って少なくとも1時間ごとに時間との5倍がレンダリングされるアクセス可能にサンプルを凍結する。 </li><li>希釈剤（33％、66％、各ステップで15分のための100％）としてメタノールと0.05％PBSTに戻って組織を再水和。 </li><li> 24時間のために溶液（0.05％PBSTで10％血清を）ブロックするに組織をブロックする。 </li><li> 48hr 5％のDMSOを含む溶液をブロッキングの1.5ミリリットルで希釈1:500の濃度でウサギ抗サイトケラチンを使用して、上皮コンテンツを識別するための一次抗体で組織をインキュベートする。 </li><li> 30分ごとに0.05％PBSTで試料を4回洗浄。 </li><li> 48hr 5％のDMSOを含む1.5ミリリットルブロッキング溶液で希釈した蛍光二次抗体（1:100ヤギ抗ウサギAlexa555）で組織をインキュベートする。 </li><li> 30分ごとに、0.05％PBSTで4回、再度サンプルを洗浄してください。 </li></ol> 4。光学投影トモグラフィ光学投影断層は最大15ミリメートル5の厚さと蛍光生体試料の高解像度3D画像を生成するために以前に開発された顕微鏡技術です。 <ol><li> sをマウントするtained 1％低融点アガロースのゲルと24時間のために100％メタノールで脱水。 48hrのためバブソリューション（1:2ベンジルアルコール、安息香酸ベンジル）で試料をクリアして、OPT 3001Mスキャナ（Bioptonics）を使用してスキャン。 </li><li>ターゲットと自家蛍光の両方をCy3フィルターセット（エキサイター545nm/30 NM、エミッタ75分の610 nm）とそれぞれGFP1フィルターセット（エキサイター425nm/40 NM、エミッタLP475 nm）を使用してキャプチャされます。 </li></ol> 5。復興と三次元定量化<ol><li> OPTが取得した角突起のセットは、浸潤アッセイの断面スライスのシリーズを作成するNReconソフトウェア（SkyScan）を使用して再構成することもできる。このソフトウェアは無償でダウンロードすることが可能です<a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> 。 </li><li> Volocityソフトウェア（パーキンエルマー社）を使用して再構成されたデータを分析。無料のデモソフトは、でダウンロードすることが可能です<a href="http://www.cellularimaging.com/products/Volocity/demo/">http://www.cellularimaging.com/products/Volocity/demo/</a> 。 </li><li>サイトケラチン染色により上皮量を定量化するために、ターゲットチャネルにおけるバックグラウンド蛍光からの干渉を低減するための最小ボクセル強度のしきい値を設定します。我々は、計測アプリケーションで使用可能なパラメータのリストを使用して9000のauで経験的にこのしきい値を定義した、我々はオリジナルのコア（自家蛍光チャンネルに区分さ）との連続性の上皮の量を定量化した。これは、私たちは異なるとの間で上皮性増殖のボリュームを定量化することを許可腫瘍サンプル。 </li></ol> 6。代表的な結果： アッセイの開発中に、52乳癌の合計は、組織病理学の広い範囲で標本を生検に使用されている。これらの&lt;10％の人が（5 / 52）少なくとも一つの実行可能な浸潤アッセイを提供するために失敗した。アッセイの失敗は、上記感染細菌または不十分な腫瘍生検材料によるものであった。したがって、アッセイのアプリケーションは、乳がんの特定のサブタイプに限定されるものではない。 典型的なアッセイ（図1）はコラーゲンの培養で20日後に固定し、その後、サイトケラチンのために染色。 Volocityソフトウェアは、オリジナルのコアと浸潤細胞の分離されたグループとの連続的な上皮バルクの両方を認識し、個別にそれぞれのボリュームを定量化することができます。 <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="図1" /> 固定および定量は、次の図1。浸潤アッセイ。アッセイは、複数の乳癌のサブタイプから得られた試​​料をサポートしています：）ER + b）のHER2 +とc）ER -。

<ol>
	<li>Lopez-Garcia, M. A., Geyer, F. C., Lacroix-Triki, M., Marchio, C., Reis-Filho, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+precursors+revisited:+molecular+features+and+progression+pathways.+Histopathology.">Breast cancer precursors revisited: molecular features and progression pathways. Histopathology.</a> 57, 171-192 (2010).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J Cell Biol. 185, 11-19 (2009).</li><li>Dabrosin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+extracellular+local+levels+of+estradiol+in+normal+breast+in+vivo+during+the+luteal+phase+of+the+menstrual+cycle.">Increased extracellular local levels of estradiol in normal breast in vivo during the luteal phase of the menstrual cycle.</a> J Endocrinol. 187, 103-108 (2005).</li><li>Alanentalo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+molecular+imaging+and+3D+quantification+within+adult+mouse+organs.">Tomographic molecular imaging and 3D quantification within adult mouse organs.</a> Nat Methods. 4, 31-33 (2007).</li><li>Sharpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Hidalgo-Carcedo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+requires+suppression+of+actomyosin+at+cell-cell+contacts+mediated+by+DDR1+and+the+cell+polarity+regulators+Par3+and+Par6.">Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6.</a> Nat Cell Biol. 13, 49-58 (2011).</li><li>Katz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+on+the+HER2+amplicon,+C35,+is+an+oncogene+in+breast+cancer+whose+actions+are+prevented+by+inhibition+of+Syk.">A gene on the HER2 amplicon, C35, is an oncogene in breast cancer whose actions are prevented by inhibition of Syk.</a> Br J Cancer. 103, 401-410 (2010).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li></ol>

縫製、JFはBioptonics OPTスキャナを製品化されたMRC技術、によって雇用されている。他の著者は、利害の抵触を宣言しません。

癌細胞株の挙動を研究するためにコラーゲンベースのアッセイは、現在広く6,7,8を使用されています。それにもかかわらず、これらのアッセイは、完全な腫瘍の複雑さとその環境に反映していない。本研究では、我々はヒト乳癌の材料は同じような方法は、ex vivoで使用できることを示す。更に、OPTは、乳がんの3D拡大を特徴付けるために有用なツールです。我々はこの技術の主な制限は、腫瘍の材料（シングルコア生検に4〜6アッセイに十分である）の可用性であることがわかった。別の制限要因は、その構造的な侵略は、アッセイの約40％でのみ発生します。これを克服するために、我々は定期的に目に見える成長とすべてのアッセイに7日目の周りの任意の治療を追加します。 OPTの使用はまた、薬物治療の結果の一括変更、ex vivoで 、検出するために将来的に拡張することができます。これは順番に、動物モデルに依存しない、より個別化がん治療を促進することができます。我々は現在、タモキシフェンex vivoでのER +の腫瘍の反応を探っています。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>完全MEGM </td><td>ロンザ</td><td> CC - 3150 </td></tr><tr><td> DMEM/F12 </td><td>シグマアルドリッチ</td><td> D8900 10X1L </td></tr><tr><td> β-エストラジオール</td><td>シグマアルドリッチ</td><td> E8875 - 250mgの</td></tr><tr><td>ウサギ抗サイトケラチン</td><td>ダコ</td><td> Z0622 </td></tr><tr><td>ヤギ抗ウサギAlexa555 </td><td>インビトロジェン</td><td> A21422 </td></tr></tbody></table>他のすべての標準試薬はSigma - Aldrichから入手した。

long-term culture of human breast cancer specimens and their analysis using optical projection tomography

乳がんは、西洋世界における死亡の主要な原因です。それはよく乳がんの広がりは、最初にローカルで、後に遠位、患者の予後における主要な因子であることが確立されている。乳癌の実験系は、通常、原発腫瘍または胸水由来の細胞株に依存しています。二つの大きな障害は、この研究の妨げになる：（i）は乳癌のいくつかの既知のサブタイプ（特に予後不良腔側Bの腫瘍は）現在の行のコレクション内で表現されていないこと、（ii）腫瘍の微小環境の影響は、通常は考慮されません。 我々は、すべてのサブタイプの文化原発性乳癌標本のテクニックを示しています。これは、腫瘍の小片が、私はクッションソフトラットのコラーゲンに埋め込まれている3次元（3D）培養系を用いることによって達成される。 2〜3週間以内に、腫瘍細胞はコラーゲンに広がり、人間tumours1で観察されたものと同様の様々な構造を形成する。オリジナルのコア内で実行可能な脂肪細胞、上皮細胞と線維芽細胞は組織学的に明らかにした。 squamoid形態と悪性上皮細胞は、周囲のコラーゲンに侵入する実証された。核多形性は有糸分裂像およびアポトーシス小体とともに、これらの細胞内で明らかであった。 我々は、培養中の腫瘍の広がりの程度を定量化するために光学投影断層撮影法（OPT）、3Dイメージング技術を、採用している。我々は腫瘍の文化のバルク量を測定するためにOPTを使用した、パラメータは、薬物療法に対する反応を評価するために、乳がん患者のネオアジュバント治療中に測定した。 ここでは、サブタイプのバイアスなしで培養ヒト乳癌腫瘍の機会を提示し、それらの生体外での広がりを定量化する。このメソッドは、元の腫瘍の薬剤感受性を定量化するために将来的に使用することができる。これは現在使用されている細胞株よりも多くの予測モデルを提供することがあります。

Watch this Scientific Journal Video about Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography at JoVE.com

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

我々は、コラーゲンベースを開発している<em&gt; in vitroで</emすべての乳癌のサブタイプのサンプルから増殖と浸潤を促進する&gt;アッセイ。光学投影断層撮影、三次元顕微鏡法は、腫瘍の拡大を視覚化し、定量化するために利用された。このアッセイは、個々の腫瘍サンプルの薬物反応を定量化するために使用されることがあります。

光学投影トモグラフィを用いたヒト乳癌検体とその分析の長期培養

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

long-term-culture-human-breast-cancer-specimens-their-analysis-using

Research

JoVE Journal

Biology

14.6K ビュー.  University of Edinburgh. 我々は、コラーゲンベースを開発している in vitroでアッセイ。光学投影断層撮影、三次元顕微鏡法は、腫瘍の拡大を視覚化し、定量化するために利用された。このアッセイは、個々の腫瘍サンプルの薬物反応を定量化するために使用されることがあります。

ビデオ: Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

広視野顕微鏡を用いて長期的なイメージング哺乳類細胞

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

心臓全体のイメージング用蛍光光学投影トモグラフィのために生まれたの正規化

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

初代ヒト卵管上皮細胞のex vivoでの文化

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

甲殻類との長期致死毒性試験アルテミアfranciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

長期成体マウスのSSCラインの導出のために文化の精原幹細胞や前駆細胞の濃縮シリアル（SSCS）

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

エクソンキャプチャと超並列配列決定することにより腫瘍標本における体細胞遺伝子変異を検出する

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

多層はゼブラフィッシュの長期シート光顕微鏡用に取り付け

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

成人マウスおよびラット心筋細胞の分離、トランスフェクション、長期培養

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...