この作品は、NIH R01 DK05823（RGWまで）、F32 DK083213（JWWまで）、F30 DK081265（ALOまで）によって、フレッドとスザンヌBiesecker小児肝センター（RGWまで）からの助成金によって賄われていた。

特記のない限り、全体の手順は室温で行われる。 1。酵素溶液の調製<ol><li> 表1及び0.22μmフィルターを用いて滅菌フィルターに応じて酵素溶液を調製する。使用時まで4℃、室温および溶液3の溶液1と溶液2°Cを保持します。単離された細胞と接触するすべてのソリューションおよび機器は、無菌でなければなりません。 </li></ol> 2。灌流装置の準備<ol><li>首相HBSSマイナスのCa 2 +灌流システム37に温めました/ Mg 2 +を 、℃注：灌流液は、それが肝臓に到達したときに、HBSSを37℃に設定し、水浴中で温めて37℃であることを確認するにはそれは血流ポンプを通過した後、それが40°C（ 図1）水循環·セットに接続されているジャケット付きガラスコンデンサーを通過します。 </li><li>に外科的滅菌70％エタノールでビーカーでオール。 </li></ol> 3。肝臓の血流<ol><li>ネンブタールの腹腔内注射（十分な麻酔を得るために必要に応じて50から100 mg / kg体重）で大人の雄Sprague-Dawleyラットを麻酔。 </li><li>プラスチック製のトレイに仰臥位で動物を置き、テープでトレイに手足を固定します。 </li><li> 70％EtOHで腹部をきれいにします。 </li><li>正中線で腹部の皮膚に小切開を行います。腹部に大きなU字型のカットを行うことによって離れて筋膜から皮膚を解剖。腹腔を露出するために、同じ形状を以下のはさみで腹部の筋肉を切り取ります。 </li><li>静かに右側に腹部内臓を移動するには、2綿棒を使用して門脈を公開します。 </li><li>門脈の下にある2つの滅菌2.0絹縫合糸を通過し、緩く接続します。 </li><li>大まかにTIを締めて、16から18ゲージの静脈カテーテルの門脈にカニューレを挿入し、場所にカテーテルを固定し前のステップ（ 図2）からEDの縫合糸。 </li><li> IVカテーテル（HBSSマイナスのCa 2 + / Mg 2 +の4ミリリットルで希釈したヘパリン5000 USP単位/ mlの1ミリリットル）を介して希釈したヘパリンを注入します。肝臓は白くする必要があります。 </li><li>灌流システムへのIVカテーテルを接続します。 + 10分20ミリリットル/分の速度でHBSSマイナスのCa 2 + / Mgを2で灌流。血液と灌流液の排水を可能にするために肝臓に劣ってIVCをトランセクト。腹腔からプールされた血液を吸引除去する。 </li><li> 0.3％コラゲナーゼ溶液（500ml HBSSプラスのCa 2 + / Mg 2 +の 2型コラゲナーゼの150 mg）と25分間灌流する灌流液を変更します。以前に解剖し、腹部フラップとそれを覆うことにより、肝臓を湿った状態に保つ。約37℃で、腹腔内温度を維持するために領域の上腹部フラップ位置の熱ランプ上のペトリ皿のカバーを置く℃に</li></ol> 4。番目の調製電子胆道系<ol><li>鉗子で腹腔からそれを持ち上げて肝臓を取り出して、冷たいリーボビッツのL-15メディアを充填した滅菌組織培養皿に置きます。 </li><li>肝臓カプセルをはがし、組織培養フードで働いて、ゆっくりと鉗子で離れて肝実質をいじめる。最終的に孤立した胆管と左まで、実質を離れていじめるを継続しながらLiebovitzのL-15メディアを充填したいくつかの料理を介して肝臓に移動します。残りの胆管が白であるのに対し、肝実質は、着色された黄褐色です。 </li><li>ペニシリン/ストレプトマイシンを10ml（10,000 IU / mlペニシリンおよび10,000μg/ mlのストレプトマイシン）を充填した50mlファルコンチューブに孤立した胆道系を配置し、15分間氷上におきます。 </li></ol> 5。ポータル線維芽細胞画分の分離<ol><li>滅菌した50mlファルコンチューブに胆管を配置し、滅菌したハサミでミンチ。 </li><li>酵素ゾルの少量を追加します。チューブにution＃1（約5ml）とは、スラリーの整合性に到達するまで、胆道系をミンチし続けています。 </li><li>滅菌したガラス瓶にスラリーを注ぐ。残りの溶液＃1をファルコンチューブを洗浄し、ガラスの瓶にそれを注ぐ。 30分間、100rpmで振とうしながら37℃でインキュベートします。 </li><li>ナイロンメッシュの単層（30ミクロンの細孔サイズ）で覆われたビーカーを介してスラリーをフィルタリングします。 </li><li>酵素溶液＃2にメッシュを洗浄することにより滅菌したガラス瓶にメッシュの上に残った組織を転送します。滅菌した15センチメートルペトリ皿にソリューションの半分（25 ml）を注入することから始めます。慎重にメッシュを反転し、中央部を汚染することなく、メッシュのエッジを保持してビーカーからメッシュを削除し、メッシュ上に残っている組織を除去するためのペトリ皿内の溶液に浸し。メッシュを破棄します。滅菌したガラス瓶にペトリ皿内の溶液を転送します。 remaのペトリ皿をすすぐ25mlのソリューション第2 iningと同じガラスの瓶に移します。 30分間、100rpmで振とうしながら37℃でインキュベートします。 </li><li>その間に、ビーカーにろ液を取り、50mlファルコンチューブに注ぐ。室温で5分間1600 rpmで（460×g）で遠心します。 </li><li>培地を吸引除去し、ソリューション＃3 25ml中にペレットを再懸濁します。 </li><li>ステップ5.5から解決策を取ると、30ミクロンのメッシュで覆われた第二滅菌ビーカーに入れ、それをフィルタリングします。 </li><li>メッシュを破棄します。室温で5分間1600 rpmでろ液を遠心分離します。吸引し、ステップ5.7で行われていたようなソリューション＃3の25ミリリットルでペレットを再懸濁します。 </li><li>ステップ5.7と5.9から2分画を組み合わせて、室温で5分間1600 rpmで再び遠心する。 </li><li>吸引は、メディアやポータル線維芽細胞培養培地を10ml（10％FBS、1％ペニシリン/ストレプトマイシン、0.3％ゲンタマイシン、および0.2％ファンギゾンを補充したDMEM/F12）でペレットを再懸濁します。 </li><li>組織培養皿に生存率およびプレートの細胞をカウントします。目的のアプリケーションに応じて、10cmの組織培養プレート当たり0.5〜5×10 6個の細胞が適切である。大人のSprague-Dawleyラットから、我々は、通常2から10000000生細胞を取得します。 </li><li>非接着性の残骸を削除するには、次の日に培地を交換してください。その後、2日毎に培地を交換してください。 </li></ol> 6。代表的な結果成体ラットの肝臓から単離された主要なポータル線維芽細胞を単離後1、3および7日（ 図3）で示されている。細胞は線維芽細胞の形態典型的に、伸長されることに注意してください。細胞は、純度を確認するために、抗エラスチン抗体（CL55041AP、Cedarlane Labsは、バーリントン、ノースカロライナ州）で染色することができます。ポータル線維芽細胞は、培養中の筋線維芽細胞分化を受けています。これは、α-平滑筋アクチン（; A2547、シグマ、セントルイス、MOα-SMA）の免疫染色によって実証することができます。 <table国境= &quot;1&quot;&gt; <tbody><tr><td rowspan="2"> 0.3％コラゲナーゼ溶液 </td><td> 2型コラゲナーゼ</td><td> 150 mgの</td></tr><tr><td>のCa 2 + / Mgの2 HBSS + </td><td> 500ミリリットル</td></tr><tr><td rowspan="7"> 解決策＃1（プロナーゼ） </td><td>ウシ血清アルブミン</td><td> 50 mgの</td></tr><tr><td>コラゲナーゼタイプ2 </td><td> 25 mgの</td></tr><tr><td>プロナーゼ</td><td> 18mgの</td></tr><tr><td>デオキシリボヌクレアーゼ</td><td> 3mgの</td></tr><tr><td> DMEM/F12 </td><td> 47.5ミリリットル</td></tr><tr><td>ペン/連鎖球菌</td><td> 1ミリリットル</td></tr><tr><td>ウシ胎仔血清</td><td> 1.5ミリリットル</td></tr><tr><td> 解決策＃2 </td><td>ウシ血清アルブミン</td><td> 50 mgの</td></tr><tr><td rowspan="6"> （ヒアルロニダーゼ） </td><td>コラゲナーゼタイプ2 </td><td> 25 mgの</td></tr><tr><td>ヒアルロニダーゼ</td><td> 22mgの</td></tr><tr><td>デオキシリボヌクレアーゼ</td><td> 3mgの</td></tr><tr><td> DMEM/F12 </td><td> 47.5ミリリットル</td></tr><tr><td>ペン/連鎖球菌</td><td> 1ミリリットル</td></tr><tr><td>ウシ胎仔血清</td><td> 1.5ミリリットル</td></tr><tr><td rowspan="2"> 解決策＃3 </td><td>デオキシリボヌクレアーゼ</td><td> 3mgの</td></tr><tr><td> RPMI培地1640 </td><td> 50ミリリットル</td></tr></tbody></table> 表1酵素ソリューションを提供しています。 <img alt="図1" src="/files/ftp_upload/3669/3669fig1.jpg" /> 図1。灌流は設定してください。A.地球温暖化列の水循環装置。灌流メディアを含むB.列。 C.コックは、ポンプの流出を制御します。 D.は、ポンプ流入します。 E.は、可変速度蠕動ポンプ。バイオクリーン洗剤でF.真空フラスコ。 G.ヒートランプ。温暖化灌流メディアのH.の水浴中。 <img alt="図2" src="/files/ftp_upload/3669/3669fig2.jpg" /> ふぃぎゅRE 2。 現場灌流します 。A. IVカテーテルは、縫合糸で固定し、活栓を介して灌流チューブに接続され、門脈に挿入されます。 B.ガラスピペットは、切離IVCの外に排出する灌流液を除去するため真空フラスコと壁吸引に接続されています。 I.の腸。 L.肝。 <img alt="図3" src="/files/ftp_upload/3669/3669fig3.jpg" /> 図3。位相コントラストおよび蛍光抗体法は、培養中のラットの門脈線維芽細胞の染色。単離後1、3、7日間培養ポータル線維芽細胞はエラスチン、α-SMAおよびDAPIに固定して染色した。

<ol>
	<li>Parola, M., Marra, F., Pinzani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblast+-+like+cells+and+liver+fibrogenesis:+Emerging+concepts+in+a+rapidly+moving+scenario.">Myofibroblast - like cells and liver fibrogenesis: Emerging concepts in a rapidly moving scenario.</a> Mol. Aspects Med. 29, 58-66 (2008).</li><li>Dranoff, J. A., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+fibroblasts:+Underappreciated+mediators+of+biliary+fibrosis.">Portal fibroblasts: Underappreciated mediators of biliary fibrosis.</a> Hepatology. 51, 1438-1444 (2010).</li><li>Tuchweber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+phenotypic+modulation+of+portal+fibroblasts+in+the+early+stages+of+cholestatic+fibrosis+in+the+rat.">Proliferation and phenotypic modulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat.</a> Lab Invest. 74, 265-278 (1996).</li><li>Beaussier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prominent+contribution+of+portal+mesenchymal+cells+to+liver+fibrosis+in+ischemic+and+obstructive+cholestatic+injuries.">Prominent contribution of portal mesenchymal cells to liver fibrosis in ischemic and obstructive cholestatic injuries.</a> Lab Invest. 87, 292-303 (2007).</li><li>Kinnman, N., Housset, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peribiliary+myofibroblasts+in+biliary+type+liver+fibrosis.">Peribiliary myofibroblasts in biliary type liver fibrosis.</a> Front Biosci. 7, d496-d503 (2002).</li><li>Kinnman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myofibroblastic+conversion+of+peribiliary+fibrogenic+cells+distinct+from+hepatic+stellate+cells+is+stimulated+by+platelet-derived+growth+factor+during+liver+fibrogenesis.">The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis.</a> Lab Invest. 83, 163-173 (2003).</li><li>Uchio, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+retinol-binding+protein-1+expression+and+modulation+during+in+vivo+and+in+vitro+myofibroblastic+differentiation+of+rat+hepatic+stellate+cells+and+portal+fibroblasts.">Cellular retinol-binding protein-1 expression and modulation during in vivo and in vitro myofibroblastic differentiation of rat hepatic stellate cells and portal fibroblasts.</a> Lab Invest. 82, 619-628 (2002).</li><li>Kruglov, E. A., Jain, D., Dranoff, J. A. <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+primary+rat+liver+fibroblasts.">Isolation of primary rat liver fibroblasts.</a> J. Investig. Med. 50, 179-184 (2002).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta+and+substrate+stiffness+regulate+portal+fibroblast+activation+in+culture.">Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture.</a> Hepatology. 46, 1246-1256 (2007).</li><li>Bosselut, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+proteomic+features+of+two+fibrogenic+liver+cell+populations:+hepatic+stellate+cells+and+portal+myofibroblasts.">Distinct proteomic features of two fibrogenic liver cell populations: hepatic stellate cells and portal myofibroblasts.</a> Proteomics. 10, 1017-1028 (2010).</li><li>Dranoff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecto-nucleoside+triphosphate+diphosphohydrolase+NTPDase2/CD39L1+is+expressed+in+a+novel+functional+compartment+within+the+liver.">The ecto-nucleoside triphosphate diphosphohydrolase NTPDase2/CD39L1 is expressed in a novel functional compartment within the liver.</a> Hepatology. 36, 1135-1144 (2002).</li></ol>

利害の衝突が宣言されません。

ポータル線維芽細胞は胆管線維症において重要な役割を果たしている。ここでは、ラット肝臓からポータル線維芽細胞を分離し、in vitroでこの細胞集団を研究するための簡単な方法を提供するためにKruglovとDranoff 8で掲載されたプロトコルの変更を説明します。 このアプローチは、プロテアーゼ消化とサイズベースのろ過を使用しています。メソッドの主な利点は、セルの比較的純粋な人口は、遺伝子発現または数日間隔離後の機能細胞の挙動の研究を可能にし、文化の中で通過せずに取得することができることである。また、この手法は、健康と病気の両方の肝臓から細胞を単離するための可能性を提供しています。 このプロトコルの中で最も重要なステップの一つは、肝実質領域の酵素消化である。成功した消化性を確保するためには、初期perfu中にその血が完全に肝臓の外に排出されていることを確認することが重要であるさらに肝臓が漂白されていることを確認することによってシオン。これを容易にするためには、灌流しながらゆっくりと肝臓をマッサージして綿棒を使用することが可能です。生細胞の収率が低いの肝実質の結果の過剰消化、下の消化が困難胆道系から肝実質を分離することになりますた。プロナーゼの準備は、異なるロット間の酵素活性の変動を持っているので、生細胞の収率が低いがある場合には、使用量の最適化が必要になることがあります。 このプロトコルは、動物の体重に比例して灌流液の体積を減少し、約10に肝灌流率が減少し、門脈をカニューレを挿入するために小さいゲージIVカテーテルを使用してマウスの肝臓や若いラットの肝臓からポータル線維芽細胞を単離するために変更することができるml /分。 α-SMAの元で明らかなように文化のポータル線維芽細胞は、10〜14日で筋線維芽細胞分化を受ける下り坂9。様々なマーカーは、肝星状フィブリン-2を含む細胞は、IL-6、エラスチン、ヌクレオシド三リン酸-2（NTPDase2）、コフィリン-1やシナプトフィジン2、6、10、11などの神経タンパク質からポータル線維芽細胞を区別するために使用されています。我々は、ポータル線維芽細胞が数日9時より後に文化を受けた後NTPDase2が失われている間エラスチンは、さらに筋線維芽細胞分化後、ポータル線維芽細胞の良いマーカーであることを発見した。したがって、我々は通常、エラスチンのための免疫蛍光染色することにより、ポータル線維芽細胞の分離を確認します。 この手法の限界はすぐにポータル線維芽細胞の分離した後、クッパー細胞と胆管細胞などの汚染のわずかな部分がある、ということです。ポータル線維芽細胞は、比較的純粋な人口（&gt; 98％）9を得た、しかし、2〜3日以内に、これらの混入細胞を脱却します。 Siの文化のポータル線維芽細胞は、組織培養プラスチック上筋線維芽細胞分化を受けるNCEは、一般的に分離7日以内に、これらの細胞を研究しています。メソッドのセクションで説明したように細胞は、10％ウシ胎児血清を含むポータル線維芽細胞培養培地で維持されます。しかし、これらの細胞は数日間、2％ウシ胎児血清を含む増殖培地中で生存します。我々は一般的に分離した後少なくとも24時間まで低血清培地を使用しないでください。ポータル線維芽細胞が筋線維芽細胞分化を受けると、それらは数回継代し、筋線維芽細胞の凍結ストックとして保持することができます。

<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> 2型コラゲナーゼ</td><td>生化学ワージントン（レイクウッド、ニュージャージー州） </td><td> LS004177 </td></tr><tr><td>プロナーゼ</td><td>ロシュ社（インディアナポリス、インディアナ州） </td><td> 70290120 </td></tr><tr><td>ヒアルロニダーゼ</td><td>シグマ（セントルイス、MO） </td><td> H3506 </td></tr><tr><td>デオキシリボヌクレアーゼ</td><td>シグマ</td><td> D4527 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen社（カリフォルニア州カールスバッド） </td><td> 11320 </td></tr><tr><td>のCa 2 + / Mgの2なしHBSS + </td><td>メディアテック（マナッサス、VA） </td><td> 21から021-CM </td></tr><tr><td>のCa 2 + / Mgの2 HBSS + </td><td>メディアテック</td><td> 21から020-CM </td></tr><tr><td>リーボビッツのL-15 </td><td>インビトロジェン</td><td> 11415 </td></tr><tr><td> RPMI培地1640 </td><td>インビトロジェン</td><td> 11875 </td></tr><tr><td>ウシ血清アルブミン</td><td>シグマ</td><td> A2153 </td></tr><tr><td>ウシ胎児血清</td><td>ジェミニバイオ（West Sacramento、カリフォルニア州） </td><td> 900から108 </td></tr><tr><td> Nitexナイロンメッシュ30μmの</td><td>ジェネシー科学（サンディエゴ、カリフォルニア州） </td><td> 57から105 </td></tr></tbody></table>

isolation of rat portal fibroblasts by in situ liver perfusion

肝線維症は、活性化線維芽細胞による細胞外マトリックスの過剰堆積によって定義されています。肝星細胞、ポータル線維芽細胞および骨髄由来線維芽細胞の1を含む肝筋線維芽細胞の複数の前駆体は、あります。肝星細胞は、最もよく研究されていますが、ポータル線維芽細胞が増加し、特に胆管線維症2で、筋線維芽細胞プールに重要な貢献者として認められています。ポータル線維芽細胞は、潜在的に胆管瘢痕3-5の初期段階で重要な役割を果たして、胆管上皮損傷に応答して増殖を受けています。ポータル線維芽細胞を単離する方法は、この細胞集団の in vitro試験で許可したポータル線維芽細胞は胆管線維症に果たす役割の理解につながる。 ポータル線維芽細胞が伸長6、7、およびLiを含む様々な技法を用いて単離されたサイズ選択8に続いて酵素消化し ​​たバージョン灌流。伸長技術に比べて消化とサイズの選択方法の利点は、細胞が培養中の通路の必要なしに研究することができるということです。ここでは、初代培養細胞の比較的純粋な人口の結果はラットの肝臓からポータル線維芽細胞を単離するためのKruglovとDranoff 8で記述されたオリジナル技法の修正版を記述します。

Watch this Scientific Journal Video about Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion at JoVE.com

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

ラット肝臓からポータル線維芽細胞を単離するための手法が説明されています。肝臓を灌流し、消化され<em&gt;その場で</em&gt;コラゲナーゼで、続いて<em&gt; ex vivoで</em肝臓スラリーと細胞の大きさの選択&gt;消化。このメソッドは、文化の通路を必要とせずにポータル線維芽細胞の純粋集団を提供する。

ラットポータル線維芽細胞の分離により、<em&gt;その場</em&gt;肝灌流

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

isolation-of-rat-portal-fibroblasts-by-in-situ-liver-perfusion

Research

JoVE Journal

Biology

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

19.2K ビュー.  The Children's Hospital of Philadelphia. ラット肝臓からポータル線維芽細胞を単離するための手法が説明されています。肝臓を灌流し、消化されその場でコラゲナーゼで、続いて ex vivoで消化。このメソッドは、文化の通路を必要とせずにポータル線維芽細胞の純粋集団を提供する。

ビデオ: Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

肝灌流による肝細胞の精製した生体肝エンドサイトーシスに

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

生物学

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

ラットのための手順<em&gt;その場で</em&gt;骨格筋収縮特性

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

蛍光活性化細胞選別（FACS）によって新鮮な組織からの正常および癌関連線維芽細胞の分離

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

二段階コラゲナーゼ灌流手順によって、ヒト肝細胞の分離

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

成体ラット心臓からの高品質心筋細胞、内皮細胞および線維芽細胞の同時単離

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心臓線維芽細胞と筋線維芽細胞の分離と特徴付け

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨格筋線維芽細胞からのエクソソームの単離と特性評価

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多パラメータ灌流制御による原発性ラット肝細胞の単離

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...