Questo lavoro è stato finanziato dal NIH R01 DK05823 (a RGW), F32 DK083213 (a JWW), F30 DK081265 (a ALO), e da una borsa di studio del Fred e Suzanne Biesecker Pediatric Liver Center (a RGW).

L&#39;intera procedura viene eseguita a temperatura ambiente a meno che diversamente specificato. 1. Preparazione di soluzioni enzimatiche <ol><li> Preparare soluzioni enzimatiche secondo la Tabella 1 e filtro sterile utilizzando un filtro di 0,22 micron. Mantenere soluzione 1 e 2 della soluzione a temperatura ambiente e la soluzione 3 a 4 ° C fino al momento dell&#39;utilizzo. Tutte le soluzioni e le attrezzature che entrano in contatto con le cellule isolate deve essere sterile. </li></ol> 2. Preparazione delle apparecchiature di perfusione <ol><li> Prime il sistema di perfusione con HBSS meno di Ca 2 + / Mg 2 +, che è stato riscaldato a 37 ° C. Nota: Per assicurare che il perfusato è 37 ° C quando arriva al fegato, HBSS è scaldata in un bagno d&#39;acqua impostata a 37 ° C. Dopo che passa attraverso la pompa di perfusione, passa attraverso un condensatore vetro incamiciato che è collegato ad una serie circolatore acqua a 40 ° C (Fig. 1). </li><li> Sterilizzare chirurgicaols in un bicchiere con il 70% di etanolo. </li></ol> 3. Perfusione del Fegato <ol><li> Anestetizzare un adulto maschio Sprague-Dawley ratto mediante iniezione intraperitoneale di Nembutal (50-100 mg / kg di peso corporeo come necessario per ottenere un&#39;adeguata anestesia). </li><li> Mettete l&#39;animale in posizione supina su un vassoio di plastica e fissare gli arti al vassoio con del nastro adesivo. </li><li> Pulire l&#39;addome con il 70% EtOH. </li><li> Fai una piccola incisione nella pelle dell&#39;addome sulla linea mediana. Dissect la pelle dalla fascia apportando U taglio sull&#39;addome. Tagliare il muscolo addominale con forbici che segue la stessa forma per esporre la cavità peritoneale. </li><li> Esporre la vena portale utilizzando 2 tamponi di cotone per spostare delicatamente il visceri addominali sul lato destro. </li><li> Passare due punti di sutura sterili in seta 2.0 sotto la vena porta e legare liberamente. </li><li> Incannulare la vena portale con un catetere di calibro 16-18 IV e fissare il catetere in posizione stringendo la liberamente tiEd suture dal passaggio precedente (Fig. 2). </li><li> Iniettare eparina diluita attraverso il catetere IV (1 ml di eparina 5000 unità USP / ml diluito con 4 ml di HBSS meno Ca 2 + / Mg 2 +). Il fegato deve sbollentare. </li><li> Collegare il catetere IV al sistema di perfusione. Perfondere con HBSS meno di Ca 2 + / Mg 2 + ad una velocità di 20 ml / min per 10 minuti. Transetto della IVC inferiore al fegato per permettere il drenaggio del sangue e perfusato. Aspirare il sangue pool dalla cavità addominale. </li><li> Cambiare il liquido di perfusione di una soluzione di collagenasi allo 0,3% (150 mg collagenasi di tipo 2 in 500 ml di HBSS più Ca 2 + / Mg 2 +) e profumato per 25 minuti. Mantenere umido il fegato coprendolo con il lembo precedentemente sezionato addominale. Coprire capsula Petri sul lembo addominale e la posizione di una lampada di calore sulla superficie di mantenere l&#39;intra-addominale temperatura a circa 37 ° C. </li></ol> 4. Preparazione del the delle vie biliari <ol><li> Rimuovere il fegato sollevandola dalla cavità addominale con una pinza e mettetelo in un piatto sterile coltura di tessuti pieni di freddo Leibovitz L-15 media. </li><li> Lavorare nel cofano coltura tissutale, rimuovere la capsula epatica e delicatamente stuzzicare il parenchima epatico a parte con una pinza. Spostare il fegato attraverso diversi piatti pieni di Liebovitz L-15 mezzi, pur continuando a prendere in giro via il parenchima, finché ha lasciato con l&#39;albero biliare isolato. Il parenchima epatico è di colore marrone, mentre l&#39;albero biliare rimanente è bianca. </li><li> Posizionare l&#39;albero biliare isolato in un tubo Falcon da 50 ml riempito con 10 ml di penicillina / streptomicina (10.000 UI / ml di penicillina e 10.000 pg / ml streptomicina); lasciare su ghiaccio per 15 minuti. </li></ol> 5. L&#39;isolamento della Frazione Fibroblast Portal <ol><li> Posizionare l&#39;albero biliare in una sterile tubo da 50 ml Falcon e tritare con le forbici sterili. </li><li> Aggiungere una piccola quantità di enzima soldistribuzione pesi # 1 nel tubo (circa 5 ml) e continuare a tritare dell&#39;albero biliare fino a raggiungere la consistenza di un impasto. </li><li> Versare l&#39;impasto in una bottiglia di vetro sterile. Lavare tubo falcon con la soluzione rimanente # 1, poi versare nella bottiglia di vetro. Incubare a 37 ° C con agitazione a 100 rpm per 30 minuti. </li><li> Filtrare la sospensione attraverso un becher coperto con un singolo strato di rete di nylon (30-micron dimensione dei pori). </li><li> Trasferire qualsiasi tessuto rimanente sulla parte superiore della maglia di una bottiglia di vetro sterile lavando la rete con soluzione enzimatica # 2. Inizia versando mezzo (25 ml) della soluzione in una capsula Petri 15 centimetri sterile. Rimuovere delicatamente la maglia dal bicchiere tenendo i bordi della maglia senza contaminare l&#39;area centrale, quindi invertendo la maglia e immersione nella soluzione nella capsula Petri per rimuovere tutto il tessuto restante sulla mesh. Gettare la maglia. Trasferire la soluzione nella capsula Petri in una bottiglia di vetro sterile. Sciacquare la piastra di Petri con la Remaesaminando la 25 ml di soluzione # 2 e trasferirlo alla bottiglia di vetro stesso. Incubare a 37 ° C con agitazione a 100 rpm per 30 minuti. </li><li> Nel frattempo, prendete il filtrato nel bicchiere e versare in una provetta da 50 ml Falcon. Centrifugare a 1600 rpm (460 x g) per 5 minuti a temperatura ambiente. </li><li> Aspirare media e risospendere il pellet in 25 ml di soluzione # 3. </li><li> Prendere la soluzione dal punto 5.5 e filtrare in un bicchiere secondo sterile coperto con maglie di 30 micron. </li><li> Gettare la maglia. Centrifugare il filtrato a 1600 rpm per 5 minuti a temperatura ambiente. Aspirare e risospendere il pellet con 25 ml di soluzione # 3 come è stato fatto nella fase 5.7. </li><li> Unire le 2 frazioni di passi 5.7 e 5.9 e centrifugare nuovamente a 1600 rpm per 5 minuti a temperatura ambiente. </li><li> Aspirare media e risospendere il pellet in 10 ml di mezzo di coltura di fibroblasti portale (DMEM/F12 supplementato con 10% FBS, 1% di penicillina / streptomicina, gentamicina 0,3%, e 0,2% fungizone). </li><li> Contare le cellule per la vitalità e la piastra in piastre di coltura dei tessuti. A seconda dell&#39;applicazione desiderata, da 0,5 a 5 x 10 6 cellule per piastra da 10 cm di coltura tissutale sono appropriati. Da un adulto Sprague-Dawley di ratto, si ottiene di solito da 2 a 10 milioni di cellule vitali. </li><li> Sostituire i terreni di coltura il giorno successivo per rimuovere i detriti non aderenti. Successivamente, sostituire terreni di coltura ogni 2 giorni. </li></ol> 6. Risultati rappresentativi Fibroblasti primari portale isolati da fegato di ratto adulto sono mostrati a 1, 3 e 7 giorni dopo l&#39;isolamento (Fig. 3). Si noti che le cellule sono allungati, con morfologia tipica di fibroblasti. Le celle possono essere colorati con anticorpi anti-elastina (CL55041AP, Cedarlane Labs, Burlington, NC) per confermare la purezza. Portale fibroblasti subiscono differenziazione miofibroblastico nella cultura. Questo può essere dimostrato mediante immunocolorazione per la α-actina del muscolo liscio (α-SMA, A2547, Sigma, St. Louis, MO). <tableborder = &quot;1&quot;&gt; <tbody><tr><td rowspan="2"> Collagenasi soluzione 0,3% </td><td> Tipo 2 collagenasi </td><td> 150 mg </td></tr><tr><td> HBSS con Ca 2 + / Mg 2 + </td><td> 500 ml </td></tr><tr><td rowspan="7"> Soluzione # 1 (Pronasi) </td><td> Albumina di siero bovino </td><td> 50 mg </td></tr><tr><td> Collagenasi di tipo 2 </td><td> 25 mg </td></tr><tr><td> Pronasi </td><td> 18 mg </td></tr><tr><td> DNasi </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> Siero fetale bovino </td><td> 1,5 ml </td></tr><tr><td> Soluzione # 2 </td><td> Albumina di siero bovino </td><td> 50 mg </td></tr><tr><td rowspan="6"> (Ialuronidasi) </td><td> Collagenasi di tipo 2 </td><td> 25 mg </td></tr><tr><td> Ialuronidasi </td><td> 22 mg </td></tr><tr><td> DNasi </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> Siero fetale bovino </td><td> 1,5 ml </td></tr><tr><td rowspan="2"> Soluzione # 3 </td><td> DNasi </td><td> 3 mg </td></tr><tr><td> RPMI media 1640 </td><td> 50 ml </td></tr></tbody></table> Tabella 1. Soluzioni enzimatiche. <img alt="Figura 1" src="/files/ftp_upload/3669/3669fig1.jpg" /> Figura 1. Perfusione istituito. A. Circolatore acqua per il riscaldamento della colonna. Colonna B. contenenti supporti di perfusione. C. Rubinetto di controllo della pompa di efflusso. D. Pump afflusso. E. a velocità variabile pompa peristaltica. F. fiasco vuoto con Bio-Clean detergente. G. calore della lampada. Acqua H. bagno per i media di perfusione riscaldamento. <img alt="Figura 2" src="/files/ftp_upload/3669/3669fig2.jpg" /> Figure 2. In perfusione situ. A. IV catetere inserito nella vena porta, fissato con sutura e collegata alla tubazione perfusione via rubinetto. B. pipetta di vetro collegato ad beuta da vuoto e di aspirazione a parete per rimuovere il liquido di perfusione drenaggio sezionato IVC. I. intestini. L. Fegato. <img alt="Figura 3" src="/files/ftp_upload/3669/3669fig3.jpg" /> Figura 3. Contrasto di fase e immunofluorescenza macchia di fibroblasti di ratto portale della cultura. Portale fibroblasti in coltura per 1, 3, e 7 giorni dopo l&#39;isolamento sono state fissate e colorate per elastina, α-SMA e DAPI.

<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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Portale fibroblasti svolgono un ruolo importante nella fibrosi biliare. Qui descriviamo una modifica del protocollo originale pubblicato da Kruglov e Dranoff 8 per isolare fibroblasti portale da fegato di ratto, fornendo un modo semplice per studiare questa popolazione di cellule in vitro. Questo approccio utilizza digestione proteasica e basato sulle dimensioni filtrazione. Il principale vantaggio del metodo è che una popolazione di cellule relativamente pura può essere ottenuta senza passaggio in coltura, consentendo lo studio dell&#39;espressione genica o comportamento cellulare funzionale diversi giorni dopo l&#39;isolamento. Questa tecnica offre anche la possibilità di isolare cellule di fegato sani e malati. Una delle fasi più critiche di questo protocollo è la digestione enzimatica del parenchima epatico. Per garantire la digestione successo, è importante per confermare che il sangue è completamente scarica dal fegato durante la prima perfusionesione facendo in modo che non vi è ancora scottatura del fegato. Per facilitare ciò, è possibile utilizzare un batuffolo di cotone per massaggiare con delicatezza il fegato mentre perfusione. Più-digestione dei risultati parenchima del fegato in una bassa resa di cellule vitali, mentre sotto-digestione renderà difficile separare il parenchima epatico dal dell&#39;albero biliare. Dal momento che le preparazioni di pronasi hanno variabilità dell&#39;attività enzimatica tra lotti differenti, l&#39;ottimizzazione della quantità utilizzata potrebbe essere necessario quando c&#39;è bassa resa di cellule vitali. Questo protocollo può essere modificato per isolare i fibroblasti portale da fegato di topo o fegato di ratto giovane utilizzando un catetere piccolo calibro IV per incannulare la vena porta, diminuendo il volume di soluzioni di perfusione in proporzione al peso degli animali, e diminuendo la velocità di perfusione del fegato a circa 10 ml / min. Portal fibroblasti in coltura subiscono differenziazione miofibroblastico in 10-14 giorni, come evidenziato da α-SMA expressione 9. Marcatori sono stati utilizzati vari per distinguere fibroblasti portale da cellule stellate epatiche, incluse fibulin-2, IL-6, elastina, nucleoside trifosfato diphosphohydrolase-2 (NTPDase2), cofilina-1 e proteine ​​neuronali quali sinaptofisina 2, 6, 10, 11 . Abbiamo scoperto che l&#39;elastina è un buon marcatore per i fibroblasti del portale, anche dopo la differenziazione miofibroblastico, mentre NTPDase2 si perde dopo fibroblasti portale sono stati sottoposti a coltura dopo diversi giorni 9. Pertanto, di solito confermano l&#39;isolamento di fibroblasti portale mediante colorazione per immunofluorescenza elastina. Il limite di questa tecnica è che, immediatamente dopo l&#39;isolamento portale fibroblasti, vi è una piccola frazione di cellule contaminanti comprese cellule di Kupffer e le cellule biliari. Fibroblasti Portal sarà troppo grande per queste cellule contaminanti nel giro di 2-3 giorni, tuttavia, producendo una popolazione relativamente puro (&gt; 98%) 9. Since portale fibroblasti in coltura subiscono differenziazione miofibroblastico su plastica di coltura di tessuti, di solito studiano queste cellule entro 7 giorni di isolamento. Le cellule vengono mantenute in mezzi di coltura di fibroblasti portale contenenti 10% di siero fetale bovino, come descritto nella sezione Metodi. Tuttavia, queste cellule sopravviveranno in terreni di coltura contenente 2% di siero fetale bovino per diversi giorni. Noi di solito non usano mezzi a basso siero fino ad almeno 24 ore dopo l&#39;isolamento. Una volta fibroblasti portale subiscono differenziazione miofibroblastico, possono essere diversi passaggi più volte e conservato come stock congelati di miofibroblasti.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Tipo 2 Collagenasi </td><td> Biochemical Worthington (Lakewood, NJ) </td><td> LS004177 </td></tr><tr><td> Pronasi </td><td> Roche (Indianapolis, IN) </td><td> 70290120 </td></tr><tr><td> Ialuronidasi </td><td> Sigma (St. Louis, MO) </td><td> H3506 </td></tr><tr><td> Deossiribonucleasi </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 senza Ca 2 + / Mg 2 + </td><td> Mediatech (Manassas, VA) </td><td> 21-021-CM </td></tr><tr><td> HBSS con 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 Media 1640 </td><td> Invitrogen </td><td> 11875 </td></tr><tr><td> Albumina di siero bovino </td><td> Sigma </td><td> A2153 </td></tr><tr><td> Siero fetale bovino </td><td> Gemini Bio (West Sacramento, CA) </td><td> 900-108 </td></tr><tr><td> Nitex Nylon Mesh 30 micron </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

Fibrosi epatica è definita dalla eccessiva deposizione di matrice extracellulare da miofibroblasti attivati. Ci sono precursori multipli di miofibroblasti epatici, tra cui le cellule stellate epatiche, fibroblasti del portale e del midollo osseo fibroblasti derivati ​​1. Cellule stellate epatiche sono stati i più studiati, ma fibroblasti portali sono sempre più riconosciute come fattori importanti per la piscina miofibroblasti, in particolare nella fibrosi biliare 2. Portale subiscono la proliferazione dei fibroblasti in risposta alla lesione epiteliale biliare, potenzialmente giocare un ruolo chiave nelle fasi iniziali della biliare cicatrici 3-5. Un metodo per isolare fibroblasti portale consentirebbe studio in vitro di questa popolazione di cellule e portare ad una maggiore comprensione del ruolo fibroblasti portale svolgere nella fibrosi biliare. Fibroblasti portale sono stati isolati utilizzando varie tecniche tra cui escrescenza 6, 7 e liperfusione ver con la digestione enzimatica seguita dalla selezione del formato 8. Il vantaggio della digestione e selezione tecnica dimensioni rispetto alla tecnica escrescenza è che le cellule possono essere studiate senza la necessità di passaggio in coltura. Qui, si descrive una versione modificata della tecnica originale descritta da Kruglov e Dranoff 8 per l&#39;isolamento di fibroblasti portale da fegato di ratto che si traduce in una popolazione relativamente pura di cellule primarie.

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

Una tecnica per isolare fibroblasti portale dal fegato di ratto è descritto. I fegati sono perfusi e digerito<em&gt; In situ</em&gt; Con collagenasi, seguita da<em&gt; Ex vivo</emDigestione&gt; dell&#39;impasto fegato e selezione dimensioni delle cellule. Questo metodo fornisce una popolazione pura di fibroblasti portale senza la necessità di passaggio in coltura.

Isolamento dei fibroblasti Portal ratto<em&gt; In situ</em&gt; Fegato perfusione

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 Views.  The Children's Hospital of Philadelphia. Una tecnica per isolare fibroblasti portale dal fegato di ratto è descritto. I fegati sono perfusi e digerito In situ Con collagenasi, seguita da Ex vivo dell'impasto fegato e selezione dimensioni delle cellule. Questo metodo fornisce una popolazione pura di fibroblasti portale senza la necessità di passaggio in coltura.

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.

In Endocitosi Fegato vivo seguita da purificazione delle cellule del fegato da parte di perfusione epatica

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 ...

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Biologia

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.

Procedure di Rat<em&gt; In situ</em&gt; Proprietà muscolatura scheletrica contrattili

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.

Isolamento di cromatina Purificazione RNA (disturbo)

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%.

Isolamento e la coltura primaria di cellule epatiche di ratto

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.

Isolamento di fibroblasti normali e tumorali associata da tessuti freschi di fluorescenza 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.

L&#39;isolamento di epatociti umani da un Two-step Collagenase perfusione procedura

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.

Isolamento simultaneo di cardiomiociti di alta qualità, cellule endoteliali e fibroblasti da un cuore di ratto adulto

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.

Isolamento e caratterizzazione di fibroblasti cardiaci adulti e miofibroblasti

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.

Isolamento e caratterizzazione di esosomi da fibroblasti muscolari scheletrici

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.

Isolamento degli epatociti primari di ratto con controllo della perfusione multiparametrica

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