我々はこのプロジェクトのさまざまな段階でのヘルプ·アドバイス·クリティカルな入力を提供したすべての仲間たちに感謝したいと思います。このビデオ物品の生産はIMIDIA（からの資金でサポートされていました<a href="http://www.imidia.org" target="_blank">http://www.imidia.org</a> ）、教育と研究のためのドイツ語省（BMBF）は糖尿病研究（DZD、のためのドイツ語センターへ<a href="http://www.dzd-ev.de" target="_blank">http://www.dzd -ev.de</a> ）技術ドレスデン大学の大学病院カール·グスタフ·カールス。本書につながる仕事は助成合意N°155005（IMIDIA）、欧州連合（EU）の第7次フレームワークプログラム（FP7/2007-2013）とEFPIAから財政的な貢献で構成されているそのうちの資源の下で革新的な医薬品·イニシアティブの共同事業からの支援を受けている企業の現物出資インチ

1。ヒト膵臓組織の凍結<ol><li>メスとピンセットで脂肪組織、血管、神経、非実質組織を削除し、ピース（〜0.5〜1センチメートルキューブ）に膵組織を切断。 </li><li> Cryomoldの中央に1膵組織片を置き、冷たいティッシュテックOCTコンパウンドで完全にそれをカバーしています。その底液体窒素で予め冷却2 - メチルブタンとスナップ凍結で覆われている瓶にCryomoldを置く。 -80℃で凍結したサンプルを保存</li></ol> 2。凍結切片の作製<ol><li> RNAの変性を避けるために、すべてのステップを通して手袋を着用してください。 </li><li>アウェイ冷たい100％エタノールおよびRNaseのクライオスタットナイフ、試料ホルダーと絵筆を清掃してください。クライオスタット内部の凍結組織を置きます。 </li><li>組織、ナイフとブラシが-20℃に到達するまで待つ待っている間にクライオスタット室の内部SuperFrostプラススラ​​イドやプレクールそれらをラベル組織は-20℃に到達するための</li><li>試料ホルダー上のティッシュ - Tek社のOCTコンパウンドを塗布し、上に凍結組織を置く。 </li><li>ティッシュテックOCT化合物が凍結されたらcryoblockをトリミングして、カミソリの刃で組織 - Tek社のOCT化合物の任意の過剰を取り除く。 </li><li>膵組織ブロックから連続4010μmのスライスの合計をカット。さらに我々は、顕微解剖プロセス中にスライス内の膵島の識別を容易にすることができ膵臓セクションの概要図面用に保存される最初と中間スライスを切ることをお勧めします。 </li><li>セクションでは、厳守しなければならないための唯一の地域を暖めるために指でスライドの裏面を（手袋で覆われて）に触れることによりSuperFrostプラススラ​​イドの中央にナイフの刃からセクションを転送します。 </li><li> -20℃クライオスタット内のセクション1-2分℃、あなたがドライアイス上に置かれたスライドの箱に入れた後、乾燥させてください。セクションを保存-80℃ </li><li>必要に応じて、紙タオルと冷たい100％エタノールでナイフの刃を清掃してください。 </li></ol> 3。凍結切片の脱水<ol><li>試薬の準備：30ミリリットル作りたての70％エタノール、30ミリリットルDEPC処理水、2x30ミリリットルの100％エタノールと50ミリリットルCellstarチューブ（ファルコン）に30mlのキシレンを注ぎ、寒さと氷の上に全ての試薬を（キシレンを除く）を保持します。 </li><li> 100％エタノールとRNaseZAPとボンネットの下に作業スペースを清掃してください。 </li><li>次のようにプロセス2凍結切片は：氷冷DEPC処理水で5月6日クイックディップで洗浄し、30秒間氷冷70％エタノールで固定し、キシレン中で4分間インキュベートし、氷冷100％エタノールで1分間ずつ2回脱水室温（25℃）、3〜5分間乾燥した空気で。凍結切片の別のペアを使用してこの手順を繰り返します。 </li><li>光や湿気から守るためにアルミホイルで包まれた50ミリリットルCellstarチューブに背中合わせに2乾燥した凍結切片を挿入します。 </li></ol> &lt;Pクラス= &quot;jove_title&quot;&gt; 4。 PALMマイクロビーム、ツァイスとのβ-細胞のレーザーマイクロダイセクション<ol><li> RNaseZAPと顕微鏡を清掃し、RoboMoverにおける接着剤キャップを取り付けます。 </li><li>フィルタセット09（励起：450から490 nmの発光&gt; 515 nm）を、AutoLPCモード、50％のレーザーエネルギー、65％のレーザー焦点、100％のレーザー速度、AutoLPCショット間5μmの距離、6μmの距離が次のように設定します。 -10100：行に、高さを働く。 </li><li>ステージに1スライドを挿入します。 </li><li>顕微鏡可視化（5倍または10倍レンズ）の下で自家β細胞を見つけます。 </li><li> 40倍のレンズに切り替えて、 &quot;フリーハンド&quot;選択ツールを使ってβ細胞を選択し、レーザーを使用してそれらを顕微解剖。 </li><li> 1 AdhesiveCapに4脱水凍結切片の組織をキャプチャします。 コメント1：1脱水凍結切片のベータ細胞を分析する時間がもたらすであろう組織切片の水分補給を避けるために10〜20分より長くするべきではありませんRNAの分解。 コメント2：チェックポイントにステージを移動させた後、目視検査によって成功した顕微解剖プロセスを確認します。 </li></ol> 5。マイクロダイセクションβ細胞濃縮組織の溶解<ol><li> RoboMoverからAdhesiveCapを削除します。 </li><li> AdhesiveCapの蓋と42でそれを逆さまインキュベート℃を30分間にピペットで10μlの抽出緩衝液（XB、PicoPure RNA単離キット、アークトゥルス）。 </li><li> 10,000 xgでスピンダウンし、ドライアイス上でライセートを置く。 RNA抽出が実行されるまで、それは、-80℃で保管してください。 </li><li>手順3を繰り返します。）〜5）。他のすべてのセクションで。 </li></ol> 6。 RNA抽出<ol><li>全1010μlの溶解物を組み合わせる</li><li> 70％エタノール（DNase処理を含む）に1:1の比率の抽出バッファー（XB）を使用して、PicoPure RNA単離キット、アルクトゥルスのプロトコルに応じて室温で働くことによってRNAの単離を進めてください。 </li></ol> 7。 RNA分析<ol><li>アジレント2100 Bioanalyser（ピコチップ社）を使用して質と量の分析のために1μlのRNAを使用します。定量的評価は、チップ上で並列にロードされた標準RNAを基準に行われます。 </li></ol>

<ol>
	<li>Bonner, R. F., Emmert-Buck, M., 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=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Suarez-Quian, C. A., Goldstein, S. R., 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=Laser+capture+microdissection+of+single+cells+from+complex+tissues.">Laser capture microdissection of single cells from complex tissues.</a> Biotechniques. 26 (2), 328-3235 (1999).</li><li>Espina, V., Wulfkuhle, J. 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=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nature Protocols. 1, 586-603 (2006).</li><li>Mikulowska-Mennis, A., Taylor, T. B., 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=High-quality+RNA+from+cells+isolated+by+laser+capture+microdissection.">High-quality RNA from cells isolated by laser capture microdissection.</a> Biotechniques. 33 (1), 176-179 (2002).</li><li>B&#246;tticher, G., Sturm, D., Ehehalt, F., Knoch, K. P., Kersting, S., Gr&#252;tzmann, R., 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=Isolation+of+Human+Islets+from+Partially+Pancreatectomized+Patients.">Isolation of Human Islets from Partially Pancreatectomized Patients.</a> J. Vis. Exp. (53), e2962 (2011).</li><li>Marselli, L., Sgroi, D. C., 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=Laser+capture+microdissection+of+human+pancreatic+beta-cells+and+RNA+preparation+for+gene+expression+profiling.">Laser capture microdissection of human pancreatic beta-cells and RNA preparation for gene expression profiling.</a> Methods in Molecular Biology. 560, 87-98 (2009).</li><li>Marselli, L., Thorne, J., 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=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clinical Endocrinology &amp; Metabolism. 93, 1046-1053 (2008).</li><li>Bottino, R., Balamurugan, A. N., 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=Response+of+human+islets+to+isolation+stress+and+the+effect+of+antioxidant+treatment.">Response of human islets to isolation stress and the effect of antioxidant treatment.</a> Diabetes. 53 (10), 2559-2568 (2004).</li><li>Abdelli, S., Ansite, J., 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=Intracellular+stress+signaling+pathways+activated+during+human+islet+preparation+and+following+acute+cytokine+exposure.">Intracellular stress signaling pathways activated during human islet preparation and following acute cytokine exposure.</a> Diabetes. 53 (11), 2815-2823 (2004).</li><li>Negi, S., Jetha, 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=Analysis+of+beta-cell+gene+expression+reveals+inflammatory+signaling+and+evidence+of+dedifferentiation+following+human+islet+isolation+and+culture.">Analysis of beta-cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isolation and culture.</a> PLoS One. 7 (1), e30415 (2012).</li></ol>

特別な利害関係は宣言されません。

我々は外科膵臓検体からヒト膵島のレーザーマイクロダイセクション（LMD）のための信頼できるアプローチについて説明します。 LMD顕微鏡が利用可能な場合、この手順は、これにより、両方の非糖尿病と2型糖尿病患者からのヒト膵島材料へのアクセスを増加させる、部分的pancreatectomiesを実行する任意の研究機関で実施されることができる。これは、膵島単離のために提供膵臓の不足与えら?...

<table border="1"><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> 2 - メチルブタン（イソペンタン） </td><td> ROTH </td><td> 3927.1 </td><td></td></tr><tr><td> AdhesiveCap（不透明、500μL） </td><td>ツァイス</td><td> 415190-9201-000 </td><td></td></tr><tr><td> Cellstarチューブ（50ml）を</td><td>グライナーバイオ1 </td><td> 210 261 </td><td>サポートスカート</td></tr><tr><td> Cellstarチューブ（50ml）を</td><td>グライナーバイオ1 </td><td> 227.261 </td><td></td></tr><tr><td>ジエチルピロカーボネート（DEPC） </td><td> SIGMA </td><td> D 5758 </td><td></td></tr><tr><td>ドライアイス</td><td></td><td></td><td></td></tr><tr><td>無水エタノール</td><td> VWR </td><td> 20821.310 </td><td></td></tr><tr><td>液体窒素</td><td></td><td></td><td></td></tr><tr><td>絵筆</td><td></td><td></td><td></td></tr><tr><td> PALMマイクロビーム</td><td>ツァイス</td><td></td><td></td></tr><tr><td>ピールウェイの埋め込み型（切り捨て）、12×12ミリメートル</td><td> ProSciTech </td><td> RR12 </td><td>トップ内部22x22のミリメートル、奥行き21ミリメートル</td></tr><tr><td>アルクトゥルスPicoPure冷凍RNA単離キット</td><td>アプライドバイオシステムズ</td><td>キット0204 </td><td></td></tr><tr><td>プラスチック製のクランプ</td><td></td><td></td><td></td></tr><tr><td>かみそり</td><td></td><td></td><td></td></tr><tr><td> RNaseフリーのDNaseセット</td><td>キアゲン</td><td> 79254 </td><td></td></tr><tr><td> RNaseZAP </td><td> SIGMA </td><td> R 2020 </td><td></td></tr><tr><td>スカルペル/外科用ブレード</td><td>テクノカット</td><td> 2800111 </td><td></td></tr><tr><td> SuperFrostプラス接着顕微鏡スライド</td><td>サーモサイエンティフィック</td><td> J1800AMN​​Z </td><td> 25x75x1.0ミリメートル</td></tr><tr><td>組織 - Tek社のOCTコンパウンド</td><td>桜</td><td> 4583または0807000022 </td><td></td></tr><tr><td>ピンセット</td><td> BRAUN </td><td> BD168R </td><td></td></tr><tr><td>キシレン</td><td> VWR </td><td> 28975.291 </td><td></td></tr></table>

improved protocol for laser microdissection of human pancreatic islets from surgical specimens

レーザーマイクロダイセクション（LMD）は実質1,2分の金額から選択した細胞や組織の回復を可能にする技術です。解剖した細胞は、トランスクリプトームやプロテオーム研究は、DNAや染色体分析評価2,3として調査、種々の用途に使用することができます。 LMDの特に困難なアプリケーションは、RNA 4の不安定性に起因して、細胞は、膵臓などのRNaseの豊富な組織から解剖される場合に特に顕著であることができ、トランスクリプトーム解析、です。標的細胞の迅速な識別と収集を可能に顕微解剖プロトコルは組織の処理時間を短くすると、その結果、RNAの保全性を確保するためには、この設定には不可欠です。 ここでは、トランスクリプトーム研究5に使用する手術標本からヒト膵β細胞を取得するためのプロトコルを記述します。約0.5〜cの膵臓の小片m 3をティッシュテックOCTコンパウンド、直ちに2 -メチルブタンチルドで凍結し、-80℃で保存しセクショニングまで、Cに埋め込 ​​ま摘出膵臓標本の健康現れるマージン、から切り出した。 10μmの厚さの四連続切片1〜2分間クライオスタット内部に乾燥したガラススライドに個別に転送-20℃設定、、、下のクライオスタットでカットし、-80℃で保存した直ちにレーザーマイクロダイセクションの手順の前に、切片を氷冷100％の2つの1分インキュベーションすることにより、氷の寒さの中に固定されたばかりの30秒間、70％エタノールを調製し、氷冷DEPC処理水で5月6日ディップで洗浄し、脱水したエタノールは4分間キシレン（組織の脱水に使用される）に続いて、組織切片を3〜5分間、その後、風乾した。重要なことは、すべての手順は、キシレン中でのインキュベーションを除いて、氷のように冷たいの試薬 ​​を用いて行った-以前に記述されたプロトコル6以上修正。 UT氷冷試薬のilizationは、ベータ細胞の内因自家蛍光の顕著な増加をもたらした、との認識を容易にした。顕微解剖のために、4つのセクションでは、各時間脱水した：2、ホイル·ラップに入れた50 mlチューブ、湿気や漂白から組織を保護するために、残りの2は直ちに顕微解剖された。この手順は、（AutoLPC）モードに突入させた自動レーザ圧接を採用PALMマイクロビーム装置（Zeiss）を用いて行った。 40-60分よりもはや不要4凍結切片からベータ細胞/小島清の完成。細胞は1 AdhesiveCapに集め、10μlの溶解緩衝液で溶解した。トランスクリプトーム解析のために、それぞれの単一のRNA試料がピコ純粋なRNA単離キット（アルクトゥルス）を用いてRNAを抽出し、10セル顕微サンプルを結合することにより得た。このプロトコルは、このように彼らの迅速かつ正確な認識とcを容易にして、ヒトβ細胞の内因自家蛍光を改善ollection。この手順のさらなる改善は、2型糖尿病に伴う変化を理解するためのより良い可能性のある影響で、表現型が異なるβ細胞の解剖を可能にするかもしれません。

図1に示すように、修正された脱水プロトコルは、以前公表されたプロトコール6に比べてβ細胞の自家蛍光の改善につながった。記述されたプロトコルを適用すると、39外科膵臓標本のそれぞれが31&#39;544&#39;704μmの3組織/膵臓標本（： - 81&#39;522&#39;153μmの3 8&#39;742&#39;390範囲）の平均のための40のシリアル凍結切片を生成するために使用された表1</strong...

Watch this Scientific Journal Video about Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens at JoVE.com

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

レーザーマイクロダイセクションでは、実質の微量から選択された細胞の回復を可能にする技術です。ここでは、トランスクリプトーム研究のために使用される手術標本からヒト膵島を取得するためのプロトコルを記述します。我々のプロトコルは、このように彼らのコレクションを容易にして、ヒトβ細胞の内因自家蛍光を向上させます。

手術標本からヒト膵島のレーザーマイクロダイセクションのために改良されたプロトコル

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

improved-protocol-for-laser-microdissection-human-pancreatic-islets

Research

JoVE Journal

Medicine

14.3K ビュー.  Paul Langerhans Institute Dresden. レーザーマイクロダイセクションでは、実質の微量から選択された細胞の回復を可能にする技術です。ここでは、トランスクリプトーム研究のために使用される手術標本からヒト膵島を取得するためのプロトコルを記述します。我々のプロトコルは、このように彼らのコレクションを容易にして、ヒトβ細胞の内因自家蛍光を向上させます。

ビデオ: Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

手術標本の腫瘍外植片を用いて新規抗がん薬の開発のための方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

医学

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

部分的に膵臓を摘出された患者からのヒト膵島の単離

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

ヒト膵臓の収集プロトコル

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

ヒト膵島の染色プロトコル

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

肝動脈再建を用いた部分同所性肝移植のラットモデルのための外科的処置

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

ジャーナルを用いたヒト網膜手術標本のトランスクリプトーム解析

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

臨床関連動物モデルおよび3D免疫化学における使用のための外科検体からのハイグレード神経膠腫細胞培養の最適化

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

子宮摘出標本から初代ヒト子宮内膜間質細胞の確立するための2つの方法

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

RNA分析のためのヒト前立腺上皮のレーザーキャプチャーマイクロダイセクション

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

レーザーのドップラー: 膵微小血管皮膚血管運動In Vivo測定のためのツール

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...