Ce travail a été financé par le NIH R01 DK05823 (à RGW), F32 DK083213 (à JWW), F30 DK081265 (à ALO), et par une subvention de la Fred et Suzanne Biesecker Centre hépatique pédiatrique (à PBE).

L&#39;ensemble de la procédure est effectuée à température ambiante à moins d&#39;indication contraire. 1. Préparation des solutions enzymatiques <ol><li> Préparer des solutions enzymatiques selon le tableau 1 et le filtre stérile à l&#39;aide d&#39;un filtre de 0,22 um. Conserver la solution une solution à 2 et à la température ambiante et la solution 3 à 4 ° C jusqu&#39;à ce moment de l&#39;emploi. Toutes les solutions et équipements entrant en contact avec des cellules isolées doivent être stériles. </li></ol> 2. Préparation de l&#39;équipement de perfusion <ol><li> Premier système de perfusion avec HBSS moins de Ca 2 + / Mg 2 +, qui a été chauffé à 37 ° C. Remarque: Afin de s&#39;assurer que le perfusat est de 37 ° C quand il arrive au niveau du foie, HBSS est réchauffé dans un bain-marie réglé à 37 ° C. Après son passage à travers la pompe de perfusion, il passe par un condenseur de verre gainé qui est connecté à un ensemble de circulation d&#39;eau à 40 ° C (Fig. 1). </li><li> Stériliser chirurgicale pourols dans un bécher avec de l&#39;éthanol 70%. </li></ol> 3. Perfusion du foie <ol><li> Anesthésier un mâle adulte Sprague-Dawley rats par injection intrapéritonéale de Nembutal (50-100 poids corporel mg / kg est nécessaire pour obtenir une anesthésie adéquate). </li><li> Placez l&#39;animal dans une position couchée sur un plateau en plastique et de fixer les membres sur le plateau avec du ruban adhésif. </li><li> Nettoyez l&#39;abdomen avec de l&#39;EtOH 70%. </li><li> Faire une petite incision dans la peau de l&#39;abdomen sur la ligne médiane. Disséquer la peau loin de l&#39;aponévrose en faisant un grand U coupe sur l&#39;abdomen. Coupez le muscle abdominal avec des ciseaux en suivant la forme même d&#39;exposer la cavité péritonéale. </li><li> Exposer la veine porte en utilisant 2 cotons-tiges pour bouger doucement les viscères abdominaux sur le côté droit. </li><li> Passer deux stériles sutures de soie 2.0 ci-dessous la veine porte et attacher lâchement. </li><li> Cathétériser la veine porte avec un cathéter jauge 16-18 IV et fixer le cathéter en place en serrant la ti lâchesutures ED dans l&#39;étape précédente (Fig. 2). </li><li> Injecter l&#39;héparine diluée à travers le cathéter IV (1 ml d&#39;unités d&#39;héparine USP 5000 / ml dilué avec 4 ml de HBSS moins de Ca 2 + / Mg 2 +). Le foie doit blanchir. </li><li> Connecter le cathéter IV le système de perfusion. Perfuser avec du HBSS moins de Ca 2 + / Mg 2 + à un débit de 20 ml / min pendant 10 minutes. Transect l&#39;IVC inférieure vers le foie pour permettre le drainage du sang et de liquide de perfusion. Aspirer le sang regroupées à partir de la cavité abdominale. </li><li> Changer le liquide de perfusion à une solution de collagénase de 0,3% (150 mg de collagénase de type 2 dans 500 ml HBSS en plus de Ca 2 + / Mg 2 +) et perfuser pendant 25 minutes. Gardez l&#39;humidité du foie en le recouvrant avec le rabat précédemment disséqué abdominale. Mettre un couvercle boîte de Pétri sur le rabat et la position abdominale d&#39;une lampe de chaleur sur la zone à maintenir la température intra-abdominale à environ 37 ° C. </li></ol> 4. Préparation de ee l&#39;arbre biliaire <ol><li> Retirer le foie en le soulevant hors de la cavité abdominale avec une pince et le placer dans une boîte de culture de tissu stérile rempli de froid Leibovitz L-15 médias. </li><li> De travail dans la hotte de culture tissulaire, décoller la capsule du foie et doucement taquiner le parenchyme hépatique en dehors avec une pince. Déplacez le foie par plusieurs plats remplis de Liebovitz de L-15 médias tout en continuant à taquiner l&#39;écart du parenchyme, jusqu&#39;à ce que finalement parti avec l&#39;arbre isolé biliaire. Le parenchyme hépatique est de couleur beige, tandis que l&#39;arbre biliaire restante est blanc. </li><li> Placez l&#39;arbre isolé biliaire dans un tube Falcon de 50 ml remplie avec 10 ml de pénicilline / streptomycine (10.000 UI / ml de pénicilline et 10.000 mg / ml de streptomycine); laisser sur la glace pendant 15 minutes. </li></ol> 5. Isolement de la fraction des fibroblastes Portail <ol><li> Placez l&#39;arbre biliaire dans un tube stérile de 50 ml faucon et émincer avec des ciseaux stériles. </li><li> Ajouter une petite quantité d&#39;enzyme solution N ° 1 dans le tube (environ 5 ml) et de continuer à émincer l&#39;arbre biliaire jusqu&#39;à ce qu&#39;il atteigne la consistance d&#39;une pâte. </li><li> Verser la boue dans une bouteille en verre stérile. Laver tube falcon avec le reste de la solution n ° 1, puis versez-le dans la bouteille en verre. Incuber à 37 ° C avec agitation à 100 tours par minute pendant 30 minutes. </li><li> Filtrer la suspension à travers un bécher recouvert d&#39;une couche unique de maillage en nylon (taille de pores de 30 microns). </li><li> Transférer tout tissu restant au-dessus de la maille à une bouteille en verre stérile par lavage de la prise avec une solution enzymatique N ° 2. Commencez par verser la moitié (25 ml) de la solution dans un récipient stérile de 15 cm de Petri. Retirer soigneusement le maillage du bécher en maintenant les bords de la maille sans contaminer la zone centrale, puis inversant le filet et le trempage dans la solution dans la boîte de Petri pour enlever tout tissu restant sur le tamis. Jeter le filet. Transférer la solution dans la boîte de Petri dans une bouteille en verre stérile. Rincer la boîte de Pétri avec la REMAYi-ning 25 ml de solution n ° 2 et de le transférer à la bouteille en verre même. Incuber à 37 ° C avec agitation à 100 tours par minute pendant 30 minutes. </li><li> Dans le même temps, prendre le filtrat dans le bécher et verser dans un tube Falcon de 50 ml. Centrifuger à 1600 rpm (460 x g) pendant 5 minutes à température ambiante. </li><li> Aspirer les médias et remettre en suspension le culot dans 25 ml de solution n ° 3. </li><li> Prenez la solution de l&#39;étape 5.5 et de la filtrer dans un deuxième bécher stérile recouvert de maille de 30 microns. </li><li> Jeter le filet. Centrifuger le filtrat à 1600 rpm pendant 5 minutes à température ambiante. Aspirer et remettre en suspension le culot avec 25 ml de solution n ° 3 comme cela a été fait à l&#39;étape 5.7. </li><li> Mélanger les 2 fractions des étapes 5.7 et 5.9 et centrifuger à nouveau à 1600 rpm pendant 5 minutes à température ambiante. </li><li> Aspirer les médias et remettre en suspension le culot dans 10 ml de milieux de culture de fibroblastes portail (DMEM/F12 supplémenté avec 10% de FBS, 1% de pénicilline / streptomycine, gentamycine 0,3%, et 0,2% fungizone). </li><li> Compter les cellules pour assurer la viabilité et la plaque dans les plats de culture de tissus. Selon l&#39;application désirée, de 0,5 à 5 x 10 6 cellules par 10 cm plaque de culture tissulaire sont appropriés. D&#39;un adulte Sprague-Dawley rats, nous obtiennent généralement de 2 à 10 millions de cellules viables. </li><li> Remplacer les milieux de culture le lendemain pour enlever les débris non-adhérent. Par la suite, remplacez les milieux de culture tous les 2 jours. </li></ol> 6. Les résultats représentatifs Fibroblastes primaires portail isolées de foie de rat adulte sont présentés à 1, 3 et 7 jours après l&#39;isolement (Fig. 3). Remarquer que les cellules sont allongées, avec la morphologie caractéristique des fibroblastes. Les cellules peuvent être colorées avec des anticorps anti-élastine (CL55041AP, Labs Cedarlane, Burlington, Caroline du Nord) pour confirmer la pureté. Fibroblastes portail subissent une différenciation myofibroblastique dans la culture. Ceci peut être démontré par immunomarquage pour l&#39;actine musculaire lisse α-(α-SMA; A2547, Sigma, St. Louis, MO). <tableborder = &quot;1&quot;&gt; <tbody><tr><td rowspan="2"> Solution de collagénase 0,3% </td><td> Collagénase de type 2 </td><td> 150 mg </td></tr><tr><td> HBSS avec Ca 2 + / Mg 2 + </td><td> 500 ml </td></tr><tr><td rowspan="7"> Solution n ° 1 (Pronase) </td><td> Sérum-albumine bovine </td><td> 50 mg </td></tr><tr><td> La collagénase de type 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> Sérum de veau fœtal </td><td> 1,5 ml </td></tr><tr><td> Solution n ° 2 </td><td> Sérum-albumine bovine </td><td> 50 mg </td></tr><tr><td rowspan="6"> (Hyaluronidase) </td><td> La collagénase de type 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> Sérum de veau fœtal </td><td> 1,5 ml </td></tr><tr><td rowspan="2"> Solution n ° 3 </td><td> DNase </td><td> 3 mg </td></tr><tr><td> Milieu RPMI 1640 </td><td> 50 ml </td></tr></tbody></table> Tableau 1. Solutions enzymatiques. <img alt="Figure 1" src="/files/ftp_upload/3669/3669fig1.jpg" /> Figure 1. Perfusion mis en place. A. Circulateur d&#39;eau pour la colonne réchauffement. La colonne B. contenant les médias de perfusion. Robinet C commander sortie de la pompe. D. Pompe entrée. E. à vitesse variable pompe péristaltique. Bouteille isotherme avec F. Bio-Clean détergent. Lampe chauffante G.. Eau H. bain pour les médias de perfusion réchauffement. <img alt="Figure 2" src="/files/ftp_upload/3669/3669fig2.jpg" /> Figure 2. En perfusion in situ. A. Cathéter intraveineux inséré dans la veine porte, fixé avec une suture et relié à la tubulure de perfusion par l&#39;intermédiaire d&#39;robinet. B. Glass pipette reliée à fiole à vide et d&#39;aspiration murale pour éliminer le liquide de perfusion s&#39;écoule de sectionnée IVC. Intestins I.. L. foie. <img alt="Figure 3" src="/files/ftp_upload/3669/3669fig3.jpg" /> Figure 3. Le contraste de phase et l&#39;immunofluorescence tache de fibroblastes portail de rat en culture. Fibroblastes cultivés pendant portail 1, 3, et 7 jours après l&#39;isolement ont été fixées et colorées de l&#39;élastine, α-SMA et DAPI.

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	<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>

Pas de conflits d&#39;intérêt déclarés.

Fibroblastes portail jouer un rôle important dans la fibrose biliaire. Ici, nous décrivons une modification du protocole original publié par Kruglov et Dranoff 8 pour isoler les fibroblastes portail de foie de rat, en fournissant un moyen simple d&#39;étudier cette population de cellules in vitro. Cette approche utilise la digestion de protéase et basée sur la taille de filtration. Le principal avantage de cette méthode est que la population est relativement pure de cellules peuvent être obtenues sans passage dans la culture, permettant l&#39;étude de l&#39;expression des gènes ou le comportement des cellules fonctionnelles plusieurs jours après l&#39;isolement. Cette technique offre également la possibilité d&#39;isoler des cellules de foie à la fois sains et malades. Une des étapes les plus critiques de ce protocole est la digestion enzymatique du parenchyme hépatique. Pour assurer une digestion réussie, il est important de confirmer que le sang est complètement évacuée du foie au cours de la première perfusion en faisant en sorte qu&#39;il n&#39;y est même blanchir du foie. Pour faciliter cela, il est possible d&#39;utiliser un coton-tige pour masser doucement le foie tout en perfusant. Au cours de digestion des résultats du parenchyme hépatique dans un faible rendement de cellules viables, tandis que la sous-digestion, il sera difficile de séparer le parenchyme hépatique à partir de l&#39;arbre biliaire. Comme les préparatifs de la pronase ont variabilité de l&#39;activité enzymatique entre les différents lots, l&#39;optimisation de la quantité utilisée peut être nécessaire quand il ya un faible rendement de cellules viables. Ce protocole peut être modifié pour isoler les fibroblastes portail de foie de souris ou de foie de rat jeune en utilisant un gabarit plus petit cathéter IV à cathétériser la veine porte, la diminution du volume des solutions de perfusion en proportion de poids de l&#39;animal, et en diminuant le débit de perfusion du foie à environ 10 ml / min. Fibroblastes portail de la culture subissent une différenciation myofibroblastique en 10-14 jours, comme en témoignent les α-SMA excompression 9. Divers marqueurs ont été utilisés pour distinguer les fibroblastes portail à partir de cellules étoilées du foie, y compris fibuline-2, IL-6, l&#39;élastine, nucléoside triphosphate diphosphohydrolase-2 (NTPDase2), cofiline-1 et des protéines neuronales telles que la synaptophysine 2, 6, 10, 11 . Nous avons constaté que l&#39;élastine est un bon marqueur pour les fibroblastes portail, même après la différenciation myofibroblastique, tandis que NTPDase2 est perdu après fibroblastes portail ont subi la culture, après plusieurs jours 9. Par conséquent, nous avons généralement de confirmer l&#39;isolement des fibroblastes portail par immunofluorescence de l&#39;élastine. La limitation de cette technique est que, immédiatement après l&#39;isolement des fibroblastes portail, il ya une petite fraction de contaminer les cellules, y compris les cellules de Kupffer et les cellules biliaires. Fibroblastes portail va dépasser ces cellules contaminantes dans les 2-3 jours, cependant, ce qui donne une population relativement pur (&gt; 98%) 9. Since fibroblastes portail de la culture subissent une différenciation myofibroblastique sur le plastique de culture tissulaire, nous avons généralement l&#39;étude de ces cellules dans les 7 jours d&#39;isolement. Les cellules sont maintenues dans le portail des milieux de culture de fibroblastes contenant 10% de sérum de veau fœtal, comme décrit dans la section des méthodes. Cependant, ces cellules vont survivre dans des milieux de croissance contenant 2% de sérum de veau fœtal pendant plusieurs jours. En général, nous n&#39;utilisons pas de faibles milieux sans sérum pendant au moins 24 heures après l&#39;isolement. Une fois les fibroblastes portail subissent une différenciation myofibroblastique, ils peuvent être repiquées à plusieurs reprises et conservées comme des stocks congelés de myofibroblastes.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Collagénase de type 2 </td><td> Biochimique Worthington (Lakewood, NJ) </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 sans Ca 2 + / Mg 2 + </td><td> Mediatech (Manassas, VA) </td><td> 21-021-CM </td></tr><tr><td> HBSS avec 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> Milieu RPMI 1640 </td><td> Invitrogen </td><td> 11875 </td></tr><tr><td> Albumine de sérum bovin </td><td> Sigma </td><td> A2153 </td></tr><tr><td> Sérum fœtal bovin </td><td> Gemini Bio (West Sacramento, Californie) </td><td> 900-108 </td></tr><tr><td> Nitex Nylon Mesh 30 um </td><td> Genesee scientifique (San Diego, CA) </td><td> 57-105 </td></tr></tbody></table>

isolation of rat portal fibroblasts by in situ liver perfusion

La fibrose du foie est défini par le dépôt excessif de matrice extracellulaire par les myofibroblastes activés. Il ya plusieurs précurseurs de myofibroblastes hépatiques, y compris les cellules étoilées du foie, des fibroblastes portail et la moelle osseuse des fibroblastes issus 1. Cellules étoilées hépatiques ont été la mieux étudiée, mais les fibroblastes du portail sont de plus en plus reconnue en tant que contributeurs importants à la piscine myofibroblastes, en particulier dans la fibrose biliaire 2. Fibroblastes portail subissent une prolifération en réponse à des lésions épithéliales biliaires, ce qui pourrait jouer un rôle clé dans les premiers stades de la cicatrisation biliaire 3-5. Une méthode d&#39;isolement des fibroblastes portail permettrait étude in vitro de cette population cellulaire et conduire à une meilleure compréhension du rôle des fibroblastes portail jouer dans la fibrose biliaire. Fibroblastes portail ont été isolés en utilisant diverses techniques, y compris l&#39;excroissance 6, 7 et liperfusion ver avec digestion enzymatique suivie par la sélection de 8 taille. L&#39;avantage de la digestion et de la technique de sélection de taille par rapport à la technique de l&#39;excroissance est que les cellules peuvent être étudiés sans la nécessité de passage dans la culture. Ici, nous présentons une version modifiée de la technique originale décrite par Kruglov et Dranoff 8 pour l&#39;isolement des fibroblastes portail de foie de rat qui se traduit par une population relativement pure de cellules primaires.

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

Une technique pour isoler les fibroblastes portail de foie de rat est décrit. Foies sont perfusés et digéré<em&gt; In situ</em&gt; Avec de la collagénase, suivie par<em&gt; Ex vivo</emLa digestion&gt; de la suspension du foie et de sélection de taille de cellules. Ce procédé fournit une population pure de fibroblastes portail sans qu&#39;il soit nécessaire pour le passage de la culture.

Isolement des fibroblastes de rat par portail<em&gt; In situ</emPerfusion du foie&gt;

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 vues.  The Children's Hospital of Philadelphia. Une technique pour isoler les fibroblastes portail de foie de rat est décrit. Foies sont perfusés et digéré In situ Avec de la collagénase, suivie par Ex vivo de la suspension du foie et de sélection de taille de cellules. Ce procédé fournit une population pure de fibroblastes portail sans qu'il soit nécessaire pour le passage de la culture.

Vidéo: 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.

Dans l&#39;endocytose hépatique in vivo suivie d&#39;une purification des cellules du foie par perfusion du foie

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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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.

Procédures pour Rat<em&gt; In situ</em&gt; Propriétés contractiles musculaires squelettiques

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.

Isolement de la chromatine par purification d&#39;ARN (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%.

L&#39;isolement et la culture primaire de cellules de foie de rat

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 des fibroblastes normales et cancéreuses associées à partir de tissus frais par tri cellulaire activé par fluorescence (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.

Isolement des hépatocytes humains par un deux-étape collagénase procédure de 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.

Isolation simultanée de cardiomyocytes de haute qualité, de cellules endothéliales et de fibroblastes d&#39;un coeur de rat adulte

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.

Isolement et caractérisation des fibroblastes cardiaques adultes et des myofibroblastes

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.

Isolement et caractérisation d’exosomes à partir de fibroblastes musculaires squelettiques

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.

Isolement des hépatocytes primaires de rat avec contrôle de perfusion multiparamétrique

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