Bu çalışma NIH R01 DK05823 (RGW için), F32 DK083213 (JWW için), F30 DK081265 (ALO) tarafından ve Fred ve Suzanne Biesecker Pediatrik Karaciğer Merkezi (RGW için) bir hibe ile finanse edildi.

Aksi belirtilmediği sürece tüm işlemi, oda sıcaklığında gerçekleştirilir. 1. Enzim Çözümler hazırlanması <ol><li> 0.22 um bir filtre kullanılarak, Tablo 1 ve steril filtre göre enzim çözümler hazırlayın. Kullanım zamanı kadar çözelti 1 ve 4 oda sıcaklığında ve solüsyon 3 azından çözeltisi 2 ° C&#39;de tutmak. Izole edilmiş hücreler ile temas durumuna gelen bütün çözeltiler ve steril bir cihaz olmalıdır. </li></ol> 2. Perfüzyon Ekipmanları hazırlanması <ol><li> HBSS eksi Ca2 + / Mg 2 asal perfüzyon sistemi +, 37 kadar ılıtıldı edildiği ° C. Not: perfüzat bu karaciğer geldiğinde, HBSS 37 ° C&#39;ye ayarlanmış su banyosunda ısıtılır 37 ° C olduğundan emin olmak için Bu perfüzyon pompa geçtikten sonra, bu 40 ° C&#39;de (Şekil 1) için bir su sirkülasyon kümesine bağlanmış olan bir cam ceketli kondenser geçer. </li><li> Için cerrahi sterilize% 70 etanol ile bir cam kapta ols&#39;ün. </li></ol> 3. Karaciğer Perfüzyon <ol><li> Nembutal intraperitoneal (yeterli anestezi elde etmek için gerekli olarak 50-100 mg / kg vücut ağırlığı) bir yetişkin erkek Sprague-Dawley sıçan uyutmak. </li><li> Plastik bir tepsi üzerinde yatar pozisyonda hayvan yerleştirin ve bant ile tepsiye bacaklarda düzeltmek. </li><li> % 70 EtOH ile karın temizleyin. </li><li> Orta hatta karın derisinde küçük bir kesi yapmak. Karın üzerinde büyük bir U-şekilli kesim yaparak uzak fasya da deriyi parçalara ayır. Periton boşluğuna ortaya çıkarmak için aynı şekil takip makas ile karın kas kesin. </li><li> Hafifçe sağ tarafa abdominal organları taşımak için 2 pamuklu çubuk kullanarak portal ven Açığa. </li><li> Portal ven altına, iki steril 2.0 ipekle Pass ve gevşek bağlar. </li><li> Bir 16-18 göstergesi IV kateter ile portal ven kanüle ve gevşek ti sıkarak yerine kateteri sabitlemekÖnceki adımda (Şekil 2) göre ed dikişlerle. </li><li> IV kateter (HBSS eksi Ca2 + / Mg + 2 ve 4 ml ile seyreltilmiş Heparin 5000 USP ünite / ml 1 ml) ile seyreltilmiş Heparin enjekte edilir. Karaciğer mazur olmalıdır. </li><li> Perfüzyon sistemine IV kateter bağlayın. + 10 dakika süreyle 20 ml / dak &#39;lık bir oranda HBSS eksi Ca2 + / Mg 2 ile perfüze. Kan ve perfüzat drenajı sağlamak için karaciğer IVC aşağı transeksiyonu. Karın boşluğundan toplanan kan aspire. </li><li> A% 0.3 kollajenaz çözeltisi (500 ml HBSS artı Ca2 + / Mg + 2 tip 2 kollajenaz 150 mg) ve 25 dakika boyunca perfüze üzere perfüzyon sıvısı değişir. Daha önce disseke karın flep ile kaplayıp karaciğer nemli tutun. Yaklaşık 37 karın içi sıcaklığını korumak için alana karın flep ve konumunu bir ısı lambası üzerinde Petri kapak koyun ° C </li></ol> 4. Th hazırlanmasıe Safra Ağacı <ol><li> Forseps ile karın boşluğundan dışarı kaldırarak karaciğer çıkarın ve soğuk Leibovitz L-15 medya dolu steril doku kültürü tabağına yerleştirin. </li><li> Karaciğer kapsül kalkmasına, doku kültürü kaput Çalışma ve yavaşça forseps dışında karaciğer parankimi kızdırmak. Nihayet izole safra yolları ile ayrılıncaya kadar, parankim uzak tease devam ederken Liebovitz L-15 medya ile dolu birkaç yemekler aracılığıyla karaciğer taşıyın. Kalan safra yolları beyaz ise karaciğer parankimi, renkli tan. </li><li> Penisilin / streptomisin ve 10 ml (10.000 IU / ml penisilin ve 10,000 ug / ml streptomisin) ile doldurulmuş bir 50 ml Falcon tüp içine izole safra yerleştirmek; 15 dakika süreyle buz üzerinde bırakın. </li></ol> 5.. Portal Fibroblast Fraksiyonu İzolasyonu <ol><li> Steril bir 50 ml falcon tüp içine safra yerleştirin ve steril makas ile kıyma. </li><li> Enzim sol küçük bir miktar eklemetüp içine ution # 1 (5 ml) ve bir bulamaç kıvamındaki ulaşıncaya kadar safra kıyma devam etmektedir. </li><li> Steril bir cam şişeye bulamaç dökün. Kalan çözüm # 1 şahinin tüp yıkayın, sonra cam şişeye dökün. 30 dakika boyunca 100 rpm&#39;de çalkalayarak 37 ° C&#39;de inkübe edilir. </li><li> Naylon örgü tek bir kat (30 mikron gözenek boyutu) kaplı bir behere aracılığıyla bulamaç filtre. </li><li> Enzim çözeltisi # 2 ile kafes yıkanarak steril bir cam şişe örgü üstünde kalan herhangi bir doku aktarın. Steril bir 15 santimetrelik bir petri çanağı içine çözeltisi yarısının (25 ml) dökülerek başlar. Dikkatle örgü ters sonra, orta alanı kirletici olmayan örgü kenarları tutarak kaptan meş kaldırmak ve örgü üzerinde kalan herhangi bir doku kaldırmak için Petri tabağına çözelti içine daldırılması. Mesh atın. Steril bir cam şişeye Petri kabındaki solüsyonu aktarın. Rema ile Petri durulayın25 ml solüsyon # 2 ve aynı cam şişeye aktarın ining. 30 dakika boyunca 100 rpm&#39;de çalkalayarak 37 ° C&#39;de inkübe edilir. </li><li> Bu arada, beherindeki süzüntü alıp 50 ml falcon tüp içine dökün. Oda sıcaklığında 5 dakika boyunca 1600 rpm&#39;de (460 x g) santrifüjlenir. </li><li> Ortamı aspire ve solüsyon 3 # 25 ml pelletini. </li><li> Adım 5.5 çözümü alın ve 30 mikron dokulu ikinci bir steril bir beher içine filtre. </li><li> Mesh atın. Oda sıcaklığında 5 dakika boyunca 1600 rpm&#39;de santrifüje süzüntü. Aspire ve adım 5,7 olarak yapıldı çözeltisi # 3 ile 25 ml pelletini. </li><li> Adımları 5.7 ve 5.9 den 2 fraksiyonlar birleştirin ve oda sıcaklığında 5 dakika boyunca 1600 rpm&#39;de tekrar santrifüjlenir. </li><li> Ortamı aspire ve portal fibroblast kültür ortamı, 10 ml (DMEM/F12% 10 FBS,% 1 penisilin / streptomisin,% 0.3 gentamisin, ve% 0.2 fungizone ile desteklenmiş) içinde pelletini. </li><li> Canlılık ve doku kültürü yemekleri plaka için hücreleri hesaplama. İstenen uygulamaya bağlı olarak, 10 cm&#39;lik bir doku kültürü plakası başına 0,5-5 x 10 hücreleri 6 uygundur. Bir yetişkin Sprague-Dawley sıçan, biz genellikle 2-10.000.000 canlı hücreler edinin. </li><li> Yapışmaz enkaz kaldırmak için ertesi gün kültür ortamı değiştirin. Bundan sonra, her 2 gün kültür ortamı değiştirin. </li></ol> 6. Temsilcisi Sonuçlar Yetişkin sıçan karaciğer izole İlköğretim portalı fibroblastlar izolasyon sonra 1, 3 ve 7 gün (Şekil 3) gösterilir. Hücreler fibroblastların morfolojisi tipik olarak uzamış dikkat edin. Hücreler saflık onaylamak için anti-elastin antikor (CL55041AP, Cedarlane Labs, Burlington, Kuzey Carolina) ile boyanmış olabilir. Portal fibroblast kültüründe miyofibroblastik farklılaşma uğrarlar. Bu α-düz kas aktin (; A2547, Sigma, St Louis, MO α-SMA) için İmmünoboyama tarafından ortaya konabilir. <tableborder = &quot;1&quot;&gt; <tbody><tr><td rowspan="2"> % 0.3 kollajenaz çözüm </td><td> Tip 2 kollajenaz </td><td> 150 mg </td></tr><tr><td> Ca2 + / Mg + 2 ile HBSS </td><td> 500 ml </td></tr><tr><td rowspan="7"> Çözüm # 1 (Pronazın) </td><td> Sığır serum albumin </td><td> 50 mg </td></tr><tr><td> Kollajenaz tip 2 </td><td> 25 mg </td></tr><tr><td> Pronazın </td><td> 18 mg </td></tr><tr><td> DNaz </td><td> 3 mg </td></tr><tr><td> DMEM/F12 </td><td> 47.5 ml </td></tr><tr><td> Pen / Strep </td><td> 1 ml </td></tr><tr><td> Fetal sığır serumu </td><td> 1.5 ml </td></tr><tr><td> Çözüm # 2 </td><td> Sığır serum albumin </td><td> 50 mg </td></tr><tr><td rowspan="6"> (Hyaluronidase) </td><td> Kollajenaz tip 2 </td><td> 25 mg </td></tr><tr><td> Hyaluronidase </td><td> 22 mg </td></tr><tr><td> DNaz </td><td> 3 mg </td></tr><tr><td> DMEM/F12 </td><td> 47.5 ml </td></tr><tr><td> Pen / Strep </td><td> 1 ml </td></tr><tr><td> Fetal sığır serumu </td><td> 1.5 ml </td></tr><tr><td rowspan="2"> Çözüm # 3 </td><td> DNaz </td><td> 3 mg </td></tr><tr><td> RPMI 1640 ortamı </td><td> 50 ml </td></tr></tbody></table> Tablo 1. Enzim çözümleri. <img alt="Şekil 1" src="/files/ftp_upload/3669/3669fig1.jpg" /> Şekil 1.. Perfüzyon kurdu. A. Isınma sütun için su sirkülatör. B. Sütun perfüzyon medya içeren. C. Musluk pompa çıkış kontrol. D. Pompa girişi. E. Değişken hızlı peristaltik pompa. Bio-Clean deterjan ile F. Vakum şişesi. G. Isı lambası. Isınma perfüzyon medya için H. Su banyosu. <img alt="Şekil 2" src="/files/ftp_upload/3669/3669fig2.jpg" /> Figuyeniden 2. in situ perfüzyon içinde. A. IV kateter sütür ile sabitlenir ve musluk aracılığıyla perfüzyon hortumu bağlı, portal ven içine yerleştirilir. B. Cam pipet kesilen IVC akıp perfüzyon sıvısına kaldırmak için termos ve duvar emme bağlı. I. Bağırsaklar. L. Karaciğer. <img alt="Şekil 3" src="/files/ftp_upload/3669/3669fig3.jpg" /> Şekil 3. Faz kontrast ve immünfloresan kültür sıçan portalı fibroblastların leke. Izolasyonu sonra 1, 3 ve 7 gün süreyle kültüre Portal fibroblastlar elastin, α-SMA ve DAPI için sabit ve boyandı.

<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>

Çıkar çatışması ilan etti.

Portal fibroblastlar safra fibrozis önemli bir rol oynamaktadır. Burada, sıçan karaciğer portal fibroblastlar tecrit in vitro bu hücre popülasyonunu incelemek için basit bir yol sağlamak için Kruglov ve Dranoff 8 tarafından yayımlanan orijinal protokolünün bir değişiklik açıklar. Bu yaklaşım, proteaz sindirim ve boyut tabanlı filtreleme kullanır. Yöntemin önemli avantajı bir hücre oldukça saf nüfusu gen ekspresyonu ya da birkaç gün sonra izole işlevsel bir hücre davranış çalışma sağlayan pasaj kültür gerek kalmadan elde edilebilir olmasıdır. Bu teknik aynı zamanda sağlıklı ve hastalıklı hem karaciğerleri hücreleri izole etmek için bir potansiyel sunmaktadır. Bu protokolün en önemli adımlardan biri, karaciğer parankiminin enzimatik sindirimi olduğunu. Başarılı bir sindirim sağlamak için, ilk perfu sırasında kan tamamen karaciğer boşaltılır onaylamak için önemlidirHatta karaciğer orada haşlama emin yaparak umulmaktadır. Bunu kolaylaştırmak için, perfüze hafifçe karaciğer masaj pamuklu çubuk kullanmak mümkündür. Canlı hücreler düşük verim karaciğer parankimi sonuçlarının Aşırı sindirim, altı sindirimi zor safra gelen karaciğer parankimi ayırmak için yapacak ise. Pronaz hazırlıkları farklı partiler, kullanılan miktarın optimizasyonu arasındaki enzimatik aktivite değişkenlik olduğundan canlı hücreler, verimleri düşük olduğunda gerekli olabilir. Bu protokol, portal ven kanüle için daha küçük bir göstergesi IV kateter kullanarak fare karaciğer ya da genç sıçan karaciğer portal fibroblastlar izole hayvan ağırlığı ile orantılı olarak perfüzyon çözümler hacmini azaltarak ve yaklaşık 10 karaciğer perfüzyon oranının azaltılması için modifiye edilebilir ml / dak. Kültür Portal fibroblastlar gibi α-SMA ex kanıtladığı, 10-14 gün içinde miyofibroblastik farklılaşma tabipression 9. Çeşitli belirteçler fibulin-2 de dahil olmak üzere karaciğer hücreleri stellat, IL-6, elastin, gelen portal fibroblastlar ayırt etmek için kullanılmış olan nükleosit trifosfat diphosphohydrolase-2 (NTPDase2), cofilin-1 ve böyle sinaptofizin 2, 6, 10, 11 gibi nöronal protein . Bu portal fibroblastlar birkaç gün sonra, kültürün 9 geçirdikten sonra NTPDase2 kaybolur ederken elastin, hatta miyofibroblastik farklılaşma sonra, portal fibroblastlann bir belirteç olduğunu bulduk. Bu nedenle, genellikle elastin için immünofloresan boyama ile portal fibroblastların izolasyonu onaylayın. Bu tekniğin kısıtlılığı hemen portalı fibroblast izolasyon sonra, Kupffer hücreleri ve safra hücreleri dahil olmak üzere hücre kirleten küçük bir bölümünü orada olmasıdır. Kapı fibroblastlar, saf bir nüfusu (&gt;% 98) 9 veren, ancak, 2-3 gün içinde bu kirletici hücreleri geçmek olacaktır. Sikültür portalı fibroblast doku kültürü plastik miyofibroblastik farklılaşma geçmesi nce, biz genellikle izolasyon 7 gün içinde bu hücrelerin çalışma. Yöntemleri bölümünde tarif edildiği gibi hücreleri,% 10 fetal sığır serumu içeren portal fibroblast kültür ortamı içinde muhafaza edilir. Bununla birlikte, bu hücrelerin birkaç gün boyunca% 2 fetal sığır serumu içeren büyüme ortamında koruyacaktır. Biz genellikle izolasyon sonra en az 24 saat kadar düşük serum medya kullanmayın. Portal fibroblastlar miyofibroblastik farklılaşma geçmesi bir kez, onlar birkaç kez pasajlandı ve miyofibroblastların dondurulmuş stok olarak tutulabilir.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Tip 2 Kollajenaz </td><td> Biyokimyasal Worthington (Lakewood, NJ) </td><td> LS004177 </td></tr><tr><td> Pronazın </td><td> Roche (Indianapolis, IN) </td><td> 70290120 </td></tr><tr><td> Hyaluronidase </td><td> Sigma (St. Louis, MO) </td><td> H3506 </td></tr><tr><td> Deoksiribonükleaz </td><td> Sigma </td><td> D4527 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen (Carlsbad, CA) </td><td> 11320 </td></tr><tr><td> Ca 2 + Mg / 2 olmadan HBSS + </td><td> Mediatech (Manassas) </td><td> 21-021-CM </td></tr><tr><td> Ca2 + / Mg + 2 ile HBSS </td><td> Mediatech </td><td> 21-020-CM </td></tr><tr><td> Leibovitz L-15 </td><td> Invitrogen </td><td> 11415 </td></tr><tr><td> Orta RPMI 1640 </td><td> Invitrogen </td><td> 11875 </td></tr><tr><td> Bovine Serum Albumin </td><td> Sigma </td><td> A2153 </td></tr><tr><td> Sığır fetüs serumu </td><td> İkizler Bio (West Sacramento, CA) </td><td> 900-108 </td></tr><tr><td> Nitex Naylon Mesh 30 mikron </td><td> Genesee Scientific (San Diego, CA) </td><td> 57-105 </td></tr></tbody></table>

isolation of rat portal fibroblasts by in situ liver perfusion

Karaciğer fıbrozda, aktive edilmiş miyofibroblastlar tarafından hücre dışı matris aşırı depozisyonu ile tanımlanır. Karaciğer yıldızsı hücreleri, portal fibroblastlar ve kemik iliğinden elde edilen fibroblast 1 de dahil olmak üzere karaciğer miyofibroblastların birden öncüleri vardır. Karaciğer yıldızsı hücreler en iyi çalışılmış oldu, ama portal fibroblastlar giderek özellikle safra fibrozis 2, myo havuzuna önemli katkı olarak kabul edilmektedir. Portal fibroblastlar potansiyel safra 3-5 skar erken dönemlerinde önemli bir rol oynayan, biliyer epitel hasarına yanıt olarak çoğalma uğrarlar. Portal fibroblastlar izole bir yöntem bu hücre popülasyonunun in vitro çalışma izin ve portal fibroblastlar safra fibrozis oynadığı rolü daha iyi anlaşılmasına yol açacaktır. Kapı fibroblastlar outgrowth 6, 7 ve Li içeren çeşitli teknikler kullanılarak izole edilmiştirboyutu seçimi 8 izledi enzimatik sindirimi ver perfüzyon. Outgrowth teknikle karşılaştırıldığında sindirim ve boyut seçimi tekniğin avantajı hücreleri kültürüne geçiş gerek kalmadan ele alınabilir olmasıdır. Burada, birincil hücre, saf bir nüfus ile sonuçlanan o sıçan karaciğer portal fibroblastların izolasyonu için Kruglov ve Dranoff 8 tarafından tarif edilen orijinal teknik değiştirilmiş bir sürümü açıklar.

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

Sıçan karaciğerinden portal fibroblastlar izole etmek için bir yöntem tarif edilmiştir. Karaciğerleri perfüze ve sindirilir<em&gt; In situ</em&gt; Kollajenaz ile, takiben<em&gt; Ex vivo</emKaraciğer bulamaç ve hücre boyutuna seçimi&gt; sindirim. Bu yöntem, kültür geçişi için gerek olmaksızın portal fibroblastlann bir saf nüfusu sağlar.

Sıçan Portal Fibroblastlar izolasyonu ile<em&gt; Yerinde</em&gt; Karaciğer Perfüzyon

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

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Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

19.2K Görüntüleme.  The Children's Hospital of Philadelphia. Sıçan karaciğerinden portal fibroblastlar izole etmek için bir yöntem tarif edilmiştir. Karaciğerleri perfüze ve sindirilir In situ Kollajenaz ile, takiben Ex vivo sindirim. Bu yöntem, kültür geçişi için gerek olmaksızın portal fibroblastlann bir saf nüfusu sağlar.

Video: 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.

Karaciğer Hücreleri Karaciğer Perfüzyon Arıtma Takip in vivo Karaciğer endositoz

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 ...

Biyoloji

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.

Rat Prosedürleri<em&gt; In situ</em&gt; İskelet Kası Kontraktil Özellikleri

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

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Klonal Genişleme CD133 + Karaciğer Kök Hücre İzolasyonu

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

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 Saflaştırma tarafından Kromatin İzolasyon (chirp)

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%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

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.

Floresans Aktif Hücre Ayırma (FACS) Taze Dokular Normal ve Kanser ilişkili Fibroblastlar İzolasyonu

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.

Bir iki aşamalı kolajenes perfüzyon prosedür ile insan Hepatositlerin İzolasyonu

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.

Bir Yetişkin Sıçan Kalpten Yüksek Kalitede Kardiyomiyosit, Endotel Hücresi ve Fibroblastların Eşzamanlı İzolasyonu

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.

Erişkin Kardiyak Fibroblastlar ve Miyofibroblastların İzolasyon ve Karakterizasyonu

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.

İskelet Kası Fibroblastlarından Eksozomların İzolasyonu ve Karakterizasyonu

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

Sıçan Portal Fibroblastlar izolasyonu ile

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