Diese Arbeit wurde vom NIH R01 DK05823 (bis RGW), F32 DK083213 (bis JWW), F30 DK081265 (zu ALO), und durch einen Zuschuss aus dem Fred und Suzanne Biesecker Pediatric Liver Center (bis RGW) gefördert.

Das gesamte Verfahren wird bei Raumtemperatur durchgeführt, wenn nicht anders angegeben. 1. Herstellung von Enzymlösungen <ol><li> Bereiten Enzymlösungen nach Tabelle 1 und Sterilfilter mit einer 0,22 um-Filter. Halten Lösung 1 und Lösung 2 bei Raumtemperatur und 3-Lösung bei 4 ° C bis zum Gebrauch. Alle Lösungen und Geräte in Kontakt mit isolierten Zellen müssen steril sein. </li></ol> 2. Herstellung von Perfusionsausrüstung <ol><li> Prime die Perfusion System mit HBSS minus Ca 2 + / Mg 2 +, hat die auf 37 ° C erwärmt Anmerkung: Um sicherzustellen, dass das Perfusat 37 ° C ist, wenn es in der Leber gelangt, HBSS in einem Wasserbad gesetzt wurden, auf 37 ° C erwärmt Nach dem Durchlaufen der Perfusionspumpe geht, geht es durch einen Glasgefäß Kondensator, der auf einen Wassergehalt Zirkulator ist auf 40 ° C (1) verbunden ist. </li><li> Sterilisieren zu chirurgischenole in einem Becherglas mit 70% Ethanol. </li></ol> 3. Perfusion der Leber <ol><li> Anesthetize einen erwachsenen männlichen Sprague-Dawley-Ratten durch eine intraperitoneale Injektion von Nembutal (50-100 mg / kg Körpergewicht wie nötig, um eine adäquate Anästhesie zu erhalten). </li><li> Platzieren Sie das Tier in Rückenlage auf einer Plastikschale und fixieren Sie die Gliedmaßen, um das Tablett mit Klebeband. </li><li> Reinigen Sie den Bauch mit 70% EtOH. </li><li> Machen Sie einen kleinen Schnitt in der Haut am Bauch in der Mittellinie. Präparieren der Haut weg von der Fascia, indem eine große U-förmigen Ausschnitt auf dem Bauch. Schneiden Sie die Bauchmuskeln mit einer Schere im Anschluss die gleiche Form, um die Bauchhöhle aussetzen. </li><li> Setzen Sie die Pfortader durch Verwendung von 2 Wattestäbchen vorsichtig bewegen Sie die Bauchorgane an der rechten Seite. </li><li> Führen Sie zwei sterile 2,0 Seidennähte unterhalb der Pfortader und locker binden. </li><li> Kanülieren die Pfortader mit einer 16-18 Gauge IV-Katheter und sichern Sie den Katheter an Ort und durch Anziehen der lose tied Nähte aus dem vorherigen Schritt (2). </li><li> Inject verdünnten Heparin durch die IV-Katheter (1 ml Heparin 5000 USP Einheiten / ml mit 4 ml HBSS minus Ca 2 + / Mg 2 + verdünnt). Die Leber sollte blanchieren. </li><li> Verbinden Sie den IV-Katheter an die Perfusion System. Perfundieren mit HBSS minus Ca 2 + / Mg 2 + mit einer Geschwindigkeit von 20 ml / min für 10 Minuten. Durchschneiden die IVC inferior in die Leber um den Abfluss von Blut und Perfusat ermöglichen. Saugen Sie gepoolten Blut aus der Bauchhöhle. </li><li> Ändern der Perfusionsflüssigkeit mit einer 0,3% Kollagenase-Lösung (150 mg Collagenase Typ 2 in 500 ml HBSS und Ca 2 + / Mg 2 +) und perfuse für 25 Minuten. Halten Sie das feuchte Leber, indem es mit dem vorher seziert Bauchlappen. Legen Sie eine Petrischale Abdeckung über dem Bauch-Klappe und die Position einer Wärmelampe über den Bereich um den intraabdominellen Temperatur bei etwa 37 ° C zu halten </li></ol> 4. Vorbereitung von the Gallenwege <ol><li> Entfernen Sie die Leber, indem Sie sie aus der Bauchhöhle mit einer Pinzette und legen Sie sie in einem sterilen Gewebekulturschale mit kaltem Leibovitz L-15-Medien gefüllt. </li><li> Arbeiten in der Gewebekultur Kapuze, ziehen Sie die Leberkapsel und sie vorsichtig auseinander Leberparenchym mit einer Pinzette. Bewegen Sie die Leber durch verschiedene Gerichte mit Liebovitz die L-15-Medien gefüllt, während weiter entfernt das Parenchym zu necken, bis schließlich mit dem isolierten Gallenwege überlassen. Das Leberparenchym ist tan gefärbt, während die restlichen Gallenwege ist weiß. </li><li> Setzen Sie den isolierten Gallenwege in ein 50 ml Falcon-Röhrchen mit 10 ml Penicillin / Streptomycin (10.000 IU / ml Penicillin und 10.000 ug / ml Streptomycin) gefüllt; verlassen auf Eis für 15 Minuten. </li></ol> 5. Die Isolierung der Fibroblasten-Portal Fraction <ol><li> Legen Sie die Gallenwege in ein steriles 50 ml Falcon-Röhrchen und Hackfleisch mit einer sterilen Schere. </li><li> Eine kleine Menge an Enzym Solution Nr. 1 in das Rohr (etwa 5 ml) und weiterhin die Gallenwege Blatt, bis sie die Konsistenz einer Aufschlämmung erreicht. </li><li> Gießen der Aufschlämmung in eine sterile Glasflaschen. Waschen Sie den Falcon-Röhrchen mit der verbleibenden Lösung Nr. 1, dann gießen Sie sie in der Glasflasche. Bei 37 ° C unter Schütteln bei 100 UpM für 30 Minuten. </li><li> Filtern der Aufschlämmung durch ein Becherglas mit einer einzelnen Schicht aus Nylon-Mesh (30 Mikron Porengröße) abgedeckt ist. </li><li> Übertragen jedes Gewebe verbleibenden auf dem Netz in einen sterilen Glasflasche durch Waschen der Eingriff mit Enzym-Lösung # 2. Starten durch Gießen Hälfte (25 ml) der Lösung in ein steriles 15 cm Petrischale. Vorsichtig das Gitter aus dem Becher durch die Ränder des Netzes ohne Kontamination der mittleren Bereich, dann invertierenden das Netz und Eintauchen in die Lösung in der Petrischale, um jegliches verbleibende Gewebe auf dem Netz zu entfernen. Entsorgen Sie die Maschen. Übertragen Sie die Lösung in der Petrischale in eine sterile Glasflasche. Spülen Sie die Petrischale mit der REMAining 25 ml Lösung Nr. 2 und überträgt es auf dem gleichen Glasflasche. Bei 37 ° C unter Schütteln bei 100 UpM für 30 Minuten. </li><li> In der Zwischenzeit nehmen Sie die Filtrat im Kolben abgemessen und in einem 50 ml-Falcon-Röhrchen. Zentrifugieren bei 1600 min (460 x g) für 5 Minuten bei Raumtemperatur. </li><li> Saugen Sie Medien und das Pellet in 25 ml Lösung # 3. </li><li> Nehmen Sie die Lösung aus Schritt 5.5 und filtern Sie sie in ein zweites steriles Becherglas mit 30-Mikron-Mesh abgedeckt. </li><li> Entsorgen Sie die Maschen. Zentrifuge das Filtrat bei 1600 rpm für 5 Minuten bei Raumtemperatur. Verwerfen und das Pellet mit 25 ml Lösung # 3, wie in Schritt 5,7 durchgeführt wurde. </li><li> Kombinieren Sie die zwei Fraktionen aus den Schritten 5.7 und 5.9 und zentrifugieren bei 1600 rpm für 5 Minuten bei Raumtemperatur. </li><li> Saugen Sie Medien und das Pellet in 10 ml-Portal Fibroblastenkultur Medien (DMEM/F12 mit 10% FBS, 1% Penicillin / Streptomycin, 0,3% Gentamycin und 0,2% Fungizon ergänzt). </li><li> Zählen von Zellen für die Lebensfähigkeit und die Platte in Zellkulturschalen. Je nach der gewünschten Anwendung, sind 0,5 bis 5 x 10 6 Zellen pro 10-cm-Gewebekulturschalen Platte angebracht. Von einem erwachsenen Sprague-Dawley-Ratten, wir in der Regel erhalten 2-10.000.000 lebensfähigen Zellen. </li><li> Ersetzen Sie die Kulturmedien am nächsten Tag an nicht-haftenden Schmutz zu entfernen. Danach ersetzen Kulturmedien alle 2 Tage. </li></ol> 6. Repräsentative Ergebnisse Primäre Fibroblasten-Portal von erwachsenen Rattenleber isoliert werden nach 1, 3 und 7 Tage nach der Isolierung (3) gezeigt. Beachten Sie, dass die Zellen gestreckt sind, mit typischen Morphologie von Fibroblasten. Die Zellen können mit Anti-Elastin-Antikörper (CL55041AP, Cedarlane Labs, Burlington, NC) gefärbt werden, um Reinheit zu bestätigen. Portal Fibroblasten unterziehen myofibroblastischer Differenzierung in der Kultur. Dies kann durch Immunfärbung für α-smooth muscle actin (; A2547, Sigma, St. Louis, MO α-SMA) nachgewiesen werden. <tableborder = &quot;1&quot;&gt; <tbody><tr><td rowspan="2"> 0,3% Collagenase-Lösung </td><td> Typ-2-Kollagenase </td><td> 150 mg </td></tr><tr><td> HBSS mit Ca 2 + / Mg 2 + </td><td> 500 ml </td></tr><tr><td rowspan="7"> Lösung 1 (Pronase) </td><td> Rinderserumalbumin </td><td> 50 mg </td></tr><tr><td> Collagenase Typ 2 </td><td> 25 mg </td></tr><tr><td> Pronase </td><td> 18 mg </td></tr><tr><td> DNase </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> Fötales Rinderserum </td><td> 1,5 ml </td></tr><tr><td> Lösung # 2 </td><td> Rinderserumalbumin </td><td> 50 mg </td></tr><tr><td rowspan="6"> (Hyaluronidase) </td><td> Collagenase Typ 2 </td><td> 25 mg </td></tr><tr><td> Hyaluronidase </td><td> 22 mg </td></tr><tr><td> DNase </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> Fötales Rinderserum </td><td> 1,5 ml </td></tr><tr><td rowspan="2"> Lösung 3 </td><td> DNase </td><td> 3 mg </td></tr><tr><td> RPMI-Medium 1640 </td><td> 50 ml </td></tr></tbody></table> Tabelle 1. Enzym-Lösungen. <img alt="1" src="/files/ftp_upload/3669/3669fig1.jpg" /> Abbildung 1. Perfusion einrichten. A. Wasser-Thermostat für Erwärmung Spalte. B. Spalte mit Perfusion Medien. C. Hahn Steuerung Pumpe Abfluss. D. Pumpenzufluss. E. Drehzahlveränderbare Schlauchpumpe. F. Isolierkanne mit Bio-Clean Reinigungsmittel. G. Wärmelampe. H. Wasserbad zum Erwärmen Perfusion Medien. <img alt="2" src="/files/ftp_upload/3669/3669fig2.jpg" /> FiguRe 2. In-situ-Perfusion. A. IV-Katheter in die Pfortader eingeführt, gesichert mit Naht und mit dem Infusionsschlauch über Hahn. B. Glas Pipette Thermosflasche und Wandabsaugung verbunden Perfusionsflüssigkeit Ablassen von durchtrennten IVC zu entfernen. I. Darm. L. Leber. <img alt="Abbildung 3" src="/files/ftp_upload/3669/3669fig3.jpg" /> Abbildung 3. Phasenkontrast und Immunfluoreszenz von Ratten-Portal Fibroblasten in Kultur zu färben. Portal Fibroblasten für 1, 3 und 7 Tage nach der Isolierung kultiviert wurden fixiert und für Elastin, α-SMA und DAPI gefärbt.

<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>

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Portal Fibroblasten spielen eine wichtige Rolle in biliären Fibrose. Hier beschreiben wir eine Modifikation des ursprünglichen Protokolls von Kruglov und Dranoff 8 zur Isolierung Portal Fibroblasten aus Rattenleber und bietet eine unkomplizierte Möglichkeit, diese Zellpopulation in vitro Studie veröffentlicht. Dieser Ansatz verwendet Proteaseverdau und Größe-basierte Filterung. Der primäre Vorteil des Verfahrens ist, dass eine relativ reine Population von Zellen ohne Passage in Kultur erhalten werden kann, so dass die Untersuchung der Genexpression oder funktionelle Verhalten der Zelle mehrere Tage nach der Isolierung. Diese Technik bietet auch das Potenzial zur Isolierung von Zellen aus sowohl gesunden und kranken Lebern. Einer der wichtigsten Schritte in diesem Protokoll ist die enzymatische Verdauung des Leberparenchyms. Um eine erfolgreiche Verdauung zu gewährleisten, ist es wichtig, um zu bestätigen, dass das Blut vollständig aus der Leber abgeleitet während der anfänglichen PerfusionSion, indem sichergestellt wird, dass es sogar Blanchieren der Leber. Um dies zu erleichtern, ist es möglich, ein Wattestäbchen verwenden, um sanft massieren, während die Leber Perfusion. Mehr als-Verdau der Leberparenchym führt zu einer niedrigen Ausbeute von lebensfähigen Zellen, während sie unter-Verdau wird es schwierig, die Leberparenchym aus der Gallenwege zu trennen. Da Zubereitungen Pronase Variabilität in der enzymatischen Aktivität zwischen verschiedenen Chargen, Optimierung der Menge verwendet werden, haben kann notwendig, wenn es geringer Ausbeute von lebensfähigen Zellen werden. Dieses Protokoll kann zum Isolieren Portal Fibroblasten aus Mausleber oder junge Rattenleber mit einem geringeren Durchmesser IV-Katheter, die Pfortader Kanülierung, Verringern des Volumens der Perfusionslösungen im Verhältnis zu Tiergewicht, und Verringern der Leber Perfusionsrate bis etwa 10 modifiziert werden ml / min. Portal Fibroblasten in Kultur zu unterziehen myofibroblastischer Differenzierung in 10-14 Tagen, wie von α-SMA ex belegtpression 9. Verschiedene Marker wurden verwendet, um Portal-Fibroblasten von hepatischen Sternzellen, einschließlich Fibulin-2, IL-6, Elastin, unterscheiden Nukleosidtriphosphat Diphosphohydrolase-2 (NTPDase2), Cofilin-1 und neuronale Proteine ​​wie Synaptophysin 2, 6, 10, 11 . Wir haben festgestellt, dass Elastin ein guter Marker für Fibroblasten-Portal ist, auch nach myofibroblastischer Differenzierung, während NTPDase2 geht verloren, nachdem Portal Fibroblasten Kultur nach einigen Tagen 9 unterzogen worden sind. Deshalb haben wir in der Regel bestätigen Isolierung von Portal-Fibroblasten durch Immunfluoreszenzfärbung für Elastin. Der Nachteil dieser Technik ist, dass unmittelbar nach Portal Fibroblasten isoliert, gibt es einen kleinen Bruchteil der kontaminierenden Zellen einschließlich Kupffer-Zellen und Gallen-Zellen. Portal Fibroblasten wird diese kontaminierenden Zellen in 2-3 Tagen zu klein wird, jedoch so dass ein relativ reine Population (&gt; 98%) 9. Since Portal Fibroblasten in Kultur myofibroblastischer Differenzierung durchlaufen auf Gewebekultur-Plastik, wir studieren diese Zellen normalerweise innerhalb von 7 Tagen der Isolation. Die Zellen werden in Portal Fibroblastenkultur Medium, das 10% fötales Rinderserum gehalten, wie in dem Verfahrensabschnitt beschrieben. Allerdings werden diese Zellen in Wachstumsmedium, enthaltend 2% fötales Rinderserum mehrere Tage überleben. Wir verwenden normalerweise keine niedrigen Serum-Medien bis spätestens 24 Stunden nach der Isolierung. Sobald Portal Fibroblasten myofibroblastischer Differenzierung durchlaufen, können sie mehrmals passagiert werden und gehalten als gefrorene Vorräte an Myofibroblasten.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Collagenase Typ 2 </td><td> Worthington Biochemical (Lakewood, New Jersey) </td><td> LS004177 </td></tr><tr><td> Pronase </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> Deoxyribonuclease </td><td> Sigma </td><td> D4527 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen (Carlsbad, CA) </td><td> 11320 </td></tr><tr><td> HBSS ohne Ca 2 + / Mg 2 + </td><td> Mediatech (Manassas, VA) </td><td> 21-021-CM </td></tr><tr><td> HBSS mit Ca 2 + / Mg 2 + </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> RPMI-Medium 1640 </td><td> Invitrogen </td><td> 11875 </td></tr><tr><td> Rinderserumalbumin </td><td> Sigma </td><td> A2153 </td></tr><tr><td> Fötalem Rinderserum </td><td> Gemini-Bio (West Sacramento, CA) </td><td> 900-108 </td></tr><tr><td> Nitex Nylon-Mesh 30 pm </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

Leberfibrose ist durch die übermäßige Ablagerung von extrazellulärer Matrix durch aktivierte Myofibroblasten definiert. Es gibt mehrere Vorläufer der hepatischen Myofibroblasten, einschließlich hepatische Sternzellen-, Portal-Fibroblasten und Knochenmark stammenden Fibroblasten ein. Hepatischen Sternzellen waren die am besten untersuchte, sondern Portal Fibroblasten werden zunehmend als wichtige Faktoren für die Myofibroblasten Pool anerkannt, insbesondere in biliären Fibrose 2. Portal Fibroblasten unterziehen Proliferation als Reaktion auf biliären Epithelzellen Verletzungen, möglicherweise eine wichtige Rolle spielen in den frühen Stadien der Gallenwege Narben 3-5. Ein Verfahren zur Isolierung Portal Fibroblasten in vitro-Studie würde dieser Zellpopulation ermöglichen und führen zu einem besseren Verständnis der Rolle Portal Fibroblasten spielen in biliären Fibrose. Portal Fibroblasten wurden isoliert unter Verwendung verschiedener Techniken wie Auswachsen 6, 7 und liver. Perfusion mit enzymatische Verdauung von Größenauswahl 8 an. Der Vorteil der Verdauung und Größe Selektionstechnik gegenüber dem Auswachsen Technik ist, dass Zellen ohne die Notwendigkeit der Passage in Kultur untersucht werden können. Hier beschreiben wir eine modifizierte Version der ursprünglichen Technik von Kruglov und Dranoff 8 zur Isolierung von Portal Fibroblasten aus Rattenleber, dass in einer relativ reinen Population von primären Zellen beschriebenen Ergebnisse.

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

Eine Technik zur Isolierung Portal Fibroblasten aus Rattenleber beschrieben. Lebern werden perfundiert und verdaut<em&gt; In-situ-</em&gt; Mit Kollagenase, gefolgt von<em&gt; Ex vivo</em&gt; Verdauung der Leber Gülle und Größe Auswahl von Zellen. Diese Methode stellt eine reine Population von Fibroblasten-Portal ohne die Notwendigkeit für den Durchgang in der Kultur.

Isolierung von Ratten-Portal Fibroblasten durch<em&gt; In-situ</em&gt; Leberperfusion

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 Aufrufe.  The Children's Hospital of Philadelphia. Eine Technik zur Isolierung Portal Fibroblasten aus Rattenleber beschrieben. Lebern werden perfundiert und verdaut In-situ- Mit Kollagenase, gefolgt von Ex vivo Verdauung der Leber Gülle und Größe Auswahl von Zellen. Diese Methode stellt eine reine Population von Fibroblasten-Portal ohne die Notwendigkeit für den Durchgang in der Kultur.

Serie: 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.

In vivo Leber Endozytose durch Reinigung von Leberzellen durch Leberperfusion Gefolgt

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 ...

Forschung

Biologie

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.

Verfahren für die Ratte<em&gt; In situ</em&gt; Skeletal Muscle kontraktilen Eigenschaften

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.

Chromatin Isolierung von RNA-Reinigung (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%.

Isolierung und Primärkultur von Ratten Leberzellen

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.

Isolation von Normal und Krebs-assoziierte Fibroblasten von Fresh Gewebe durch 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 ...

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.

Isolierung von humanen Hepatozyten durch eine Zwei-Schritt-Verfahren Collagenase Perfusion

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.

Gleichzeitige Isolierung von hochwertigen Kardiomyozyten, Endothelzellen und Fibroblasten aus einem erwachsenen Rattenherz

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.

Isolierung und Charakterisierung von adulten Herz-Fibroblasten und Myofibroblasten

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.

Isolierung und Charakterisierung von Exosomen aus Skelettmuskelfibroblasten

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.

Isolierung primärer Rattenhepatozyten mit Multiparameter-Perfusionskontrolle

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