Les auteurs sont reconnaissants à la pathologie moléculaire et imagerie de base du Centre NIDDK UPenn d&#39;études moléculaires dans des maladies digestives et hépatiques (P30 DK50306) pour l&#39;aide à l&#39;imagerie. Ce travail a été soutenu par des subventions de la National Institutes of Health (R01 DK-092111) et de Fred et Suzanne Biesecker pédiatrique foie Center (à PBE) et par une bourse de recherche sur les maladies du foie et de l&#39;éducation de la petite enfance réseau (à SK).

Toute la procédure est effectuée à la température ambiante, sauf indication contraire. Tous les travaux des animaux doit être effectué dans des conditions humaines en vertu d&#39;un protocole approuvé par le Comité institutionnel de protection des locaux et l&#39;utilisation des animaux (IACUC). 1. Préparation de l&#39;équipement et Solutions <ol><li> Mettre en place la table chirurgicale de la souris à proximité de la hotte de culture de tissu et l&#39;incubateur (Figure 1A). Remarque: L&#39;équipement nécessaire comprend un microscope de dissection, une source de lumière, de 12,5 cm de long ciseaux iris droites, 6 pouces pince courbe en dents de scie non avec des pointes fines, et 4 pouces de pince dents de scie avec des pointes courbes (figure 1B). </li><li> Placez instruments stérilisés dans un bécher en verre de 70% d&#39;alcool sur la table d&#39;opération. </li><li> Remplir trois 60 mm boîtes de Pétri stériles avec les médias de l&#39;isolement stérile (tableau 1); placer deux sur la table chirurgicale et un dans le capot, le tout sur la glace. </li><li> Placez collagène de queue de rat,stérile 10x tampon phosphate salin (PBS), de l&#39;eau stérile, NaOH stérile 1 et biliaire cellules épithéliales (BEC) des médias (tableau 1) dans la hotte de culture de tissus. Remarque: Le collagène doit être sur la glace. </li></ol> 2. Cholagogue Isolement et culture <ol><li> Euthanasier les souris néonatale entre 0-3 jours de la vie, par exposition au dioxyde de carbone jusqu&#39;à inconscient suivie par dislocation cervicale, conformément aux lignes directrices du IACUC (figure 1C). Remarque: les souris plus âgées peuvent être utilisés en fonction des besoins expérimentaux spécifiques. </li><li> Placer l&#39;animal en décubitus dorsal et une petite incision horizontale traversant la ligne médiane dans la région du bassin. Étendre cette incision latéralement et vers le haut des deux côtés de l&#39;abdomen, en formant un rabat en forme d&#39;U. Tournez rabat pour exposer les organes abdominaux. Une fois que l&#39;abdomen est ouvert, déplacer soigneusement les intestins hors de la cavité abdominale vers la droite de l&#39;animal pour une meilleure visualisation du foie et de la voie biliaire principale. Remarque: il peut être nécessaire d&#39;étendre les incisions latérales dans la cage thoracique d&#39;obtenir une meilleure identification du foie. </li><li> Utilisation du microscope à dissection, d&#39;identifier la voie biliaire, du foie et des intestins. Identifier le canal cholédoque premier (figure 1D), en utilisant la prudence, car les structures sont très délicates. Avec les pinces dentelées et non dentelées, méticuleusement propre et enlever le tissu conjonctif y compris la graisse autour de la bile. Utiliser une pince pour maintenir le conduit et l&#39;autre pour le nettoyage. </li><li> Utiliser une technique similaire pour nettoyer la vésicule biliaire. </li><li> Placer les conduits nettoyés et la vésicule biliaire dans le premier plat de milieux d&#39;isolement sur la glace. Massez doucement les structures avec les forceps non dentelés tandis que dans les médias pour éliminer davantage le tissu conjonctif et enlever la bile de la vésicule biliaire. </li><li> Transférer les conduits et la vésicule biliaire à la deuxième plat de milieux d&#39;isolement. </li><li> Après isolement de conduites et de la vésicule biliaire à partir de 5 animaux, transférer lepièces à la troisième boîte de Pétri, dans la hotte. Remarque: si les cellules doivent être isolé à partir de plus de cinq animaux, faire en lots, en utilisant des solutions fraîches pour chaque groupe de 5 animaux. </li><li> Préparer des gels de collagène épais dans la hotte de culture de tissus: Remarque: tous les plats de taille peut être utilisé. Une boîte de culture de 60 mm avec un volume final de 3 ml de collagène pour chaque groupe de 5 nouveau-nés fonctionne bien. Ce doit être préparé au moment de l&#39;intégration conduits fraîchement isolés. <ol><li> Placer rat tail collagène, 10x stérile saline tamponnée au phosphate (PBS 10x), dH 2 O stérile, et N NaOH stérile sur de la glace 1. </li><li> Calculer les volumes de dH 2 0, 10x PBS, NaOH, et le collagène nécessaire pour des gels ayant une concentration finale de 2 mg / ml de collagène dans du PBS 1x, en utilisant un volume de 1 N de NaOH égale à 0,023 fois le volume de collagène ajoutée. Remarque: Si autre source de collagène est utilisé, suivre les instructions du fabricant pour la neutralisation et la formation de gel. </li><li> Mélanger dH 2 0, 10x PBS, et 1 N NaOH, puis ajouter le collagène et pipette bien pour assurer un mélange homogène. </li></ol></li><li> Une fois que la solution de collagène est épaissie à une consistance de gel à la température ambiante, immédiatement incorporer les conduits dans le collagène. </li><li> Placer à l&#39;étuve à 37 ° C, 5% CO 2 pendant 30 minutes pour permettre au collagène de se solidifier. </li><li> Une fois gel a solidifié (couleur passe de transparent à blanc), superposition avec 3,5 ml de milieu BEC (tableau 1) et incuber à 37 ° C, 5% de CO 2. </li><li> Retirez le support BEC 3 fois par semaine et le remplacer par 3,5 ml de milieu frais. </li></ol> 3. Cholangiocytes à isolation primaire du collagène épais Gels Remarque: dans les 3 semaines, feuilles de cholangiocytes (cellules avec de gros noyaux s&#39;étendant directement à partir de conduits intégrés) seront visibles dans le collagène d&#39;épaisseur. Si il ya un grand nombre de fibroblastes dans le plat, il doit être jeté. Si quelques régions du plat ontcroissance de fibroblastes, ceux-ci peut être découpé avec un scalpel et enlevée avant le fractionnement des cholangiocytes. <ol><li> Préparer minces gels de collagène: <ol><li> Diluer le collagène à une concentration de 1 mg / ml avec du PBS stérile. Remarque: utiliser 1 ml pour 100 plaque mm; ajuster en conséquence pour d&#39;autres plats de taille. </li><li> Fais la solution de collagène uniformément avec une spatule de cellule (grands plats) ou de la pointe de la pipette (petits plats), puis laisser à température ambiante pendant 10 min. </li><li> Couvrir la plaque avec DMEM/F12 et incuber à 37 ° C, 5% de CO2 pendant 10 min. </li><li> Retirer la plaque de l&#39;incubateur et laver avec du PBS 1X. Aspirer immédiatement 1x PBS. </li><li> Manteau avec une seconde couche mince de collagène, d&#39;étalement par rotation de la plaque ou plaque de placer sur un agitateur. Incuber à 37 ° C, 5% de CO2 pendant 10 min. </li><li> Après collagène est solidifié, la plaque de couverture avec DMEM/F12 et incuber à 37 ° C, 5% de CO2 pendant 30 min. </li></ol></li><li> Diviser les cellules de egels ick de collagène: <ol><li> Avec une spatule de culture cellulaire ou d&#39;un grattoir de cellules, gratter doucement gel de collagène de la plaque de sorte qu&#39;elle est flottant. </li><li> Préparer une solution de collagénase en diluant de type XI collagénase (voir la liste des matériaux) à 10 mg / ml avec DMEM/F12. Mélangez bien et filtre stérile. </li><li> Ajouter 1 ml de solution de collagénase (ci-dessus) par boîte de 60 mm de cellules en collagène d&#39;épaisseur. Ne retirez pas le support BEC (qui devrait être de 3 ml). Incuber plat pendant 30 min à 37 ° C, 5% CO 2. </li><li> Retirer la solution contenant des cellules, et placer dans 15 ml de tube conique. </li><li> Isoler à 2200 g pendant 5 min. Jeter le surnageant et remettre suspendre le culot dans un volume similaire de DMEM/F12. </li><li> Spin nouveau solution à 2200 g pendant 5 min. Retirer le surnageant. </li><li> Remettre en suspension le culot dans 3 ml de trypsine à 0,5%, et les incuber à 37 ° C, 5% CO 2 pendant 5 min. Après incubation, la pipette la solution de haut en bas pour briser les feuilles de cholangiocytes. Ajouter 5 ml de BEC médias unesolution de filage ème à 1500 g pendant 5 min. </li><li> Retirer le surnageant et remettre le culot en DMEM/F12, puis tourner solution à 1500 g pendant 5 min. </li><li> Retirer le surnageant et re suspendre le culot dans les médias BEC (tableau 1). Cellules de la plaque sur faites précédemment minces gels de collagène. </li></ol></li></ol> 4. Passage et le stockage des cellules Note: les cellules minces sur des gels de collagène peuvent être divisées selon les besoins. <ol><li> Laver les plaques avec du PBS stérile avant l&#39;incubation dans 0,25% de trypsine à 37 ° C pendant 10 à 15 min. </li><li> Neutraliser avec un volume égal de milieu BEC, et un culot à 1500 g pendant 5 min. Rincer culot avec DMEM/F12, alors culot à 1500 g pendant 5 min. </li><li> Resuspendre les cellules dans des milieux BEC pour la distribution de collagène revêtu des boîtes de culture cellulaire. Remarque: En variante, les cellules peuvent être remises en suspension dans un support de stockage et stockées dans un congélateur C -80 ° ou de l&#39;azote liquide (tableau 1). </li></ol>

<ol>
	<li>Paradis, K., Sharp, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+duct-like+structure+formation+after+isolation+of+bile+ductular+cells+from+a+murine+model.">In vitro duct-like structure formation after isolation of bile ductular cells from a murine model.</a> J. Lab. Clin. Med. 113 (6), 689-694 (1989).</li><li>Vroman, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+polarized+primary+cultures+of+rat+intrahepatic+bile+duct+epithelial+cells.">Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells.</a> Lab. Invest. 74 (1), 303-313 (1996).</li><li>Kumar, U., Jordan, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+biliary+epithelial+cells+from+the+biliary+tract+fraction+of+normal+rats.">Isolation and culture of biliary epithelial cells from the biliary tract fraction of normal rats.</a> Liver. 6 (6), 369-378 (1986).</li><li>Ishii, M., Vromen, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+morphologic+characterization+of+bile+duct+epithelial+cells+from+normal+rat+liver.">Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver.</a> Gastroenterology. 97 (5), 1236-1247 (1989).</li><li>Strazzabosco, M., Fabris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+bile+ducts:+Essentials+for+the+clinical+hepatologist.">Development of the bile ducts: Essentials for the clinical hepatologist.</a> J. Hepatol. 56 (5), 1159-1170 (2012).</li><li>Carpentier, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+ductal+plate+cells+give+rise+to+cholangiocytes,+periportal+hepatocytes,+and+adult+liver+progenitor+cells.">Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells.</a> Gastroenterology. 141 (4), 1432-1438 (2011).</li><li>Cho, W. K., Mennon, A., Boyer, J. L. <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+functional+polarized+bile+duct+units+from+mouse+liver.">Isolation of functional polarized bile duct units from mouse liver.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 280 (2), (2001).</li><li>Karjoo, S., Hand, N. J., Loarca, L., Russo, P. A., Friedman, J. R., 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=Extra-hepatic+cholangiocyte+cilia+are+abnormal+in+biliary+atresia.">Extra-hepatic cholangiocyte cilia are abnormal in biliary atresia.</a> J. Pediatr. Gastroenterol. Nutr. 57 (1), 96-101 (2013).</li><li>Sutton, M. E., op den Dries, S., Koster, M. H., Lisman, T., Gouw, A. S., Porte, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+human+extrahepatic+biliary+epithelium:+the+peribiliary+glands+as+progenitor+cell+compartment.">Regeneration of human extrahepatic biliary epithelium: the peribiliary glands as progenitor cell compartment.</a> Liver Int. 32 (4), 554-559 (2012).</li><li>Cardinale, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+stem/progenitor+cells+in+human+biliary+tree+give+rise+to+hepatocytes,+cholangiocytes,+and+pancreatic+islets.">Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets.</a> Hepatology. 54 (6), 2159-2172 (2011).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun conflit d&#39;intérêts.

Décrite ici est une technique pour isoler cholangiocytes pures à partir des voies biliaires extra-hépatiques de souris de souris de tous les âges, y compris les nouveau-nés. La technique offre l&#39;avantage que cholangiocytes extra-hépatiques peuvent être étudiés séparément cholangiocytes intra-hépatiques, et peut faciliter les études pour identifier les principales différences entre ces populations de cellules. Nous avons récemment publié une étude démontrant diminué cils dans cholangiocytes extra-h...

L&#39;origine et le développement de cholangiocytes intra et extra-hépatiques sont nettement différents. Le foie se développe à partir d&#39;un diverticule de l&#39;intestin antérieur endoderme ventral 5. La région caudale du diverticule forme l&#39;arbre biliaire extra-hépatique, tandis que la région crânienne génère l&#39;arbre biliaire intrahépatique 5. Cholangiocytes de conduits intrahépatiques sont dérivées de cellules progénitrices autour de la plaque canalaire dans les régions péri portail 6. Ces cellules ont la capacité de se différencier en hépatocytes ou soit des cholangiocytes 6. Cela a d&#39;importantes implications cliniques étant donné que cholangiopathies peuvent cibler spécifiquement une catégorie de cholangiocytes. Par exemple, l&#39;atrésie biliaire affecte d&#39;abord les canaux extra-hépatiques du nouveau-né, alors que le syndrome d&#39;Alagille affecte voies biliaires intrahépatiques. Il existe des descriptions multiples de l&#39;isolement des cholangiocytes intra-hépatiques et les unités de la voie biliaire à partir des souris et des rats. Dansinvestigateurs ont isolé des cellules individuelles par l&#39;isolement de la gaine et l&#39;excroissance subséquente, ou par digestion du foie et de la tirer vers le bas anticorps cholangiocytes exprimant des marqueurs de surface cellulaire spécifiques 4.1. Les unités des canaux biliaires fonctionnels et polarisés ont été isolées par digestion du foie et de la filtration de la taille 7. Biliaires unités de conduits sont capables de répondre à des stimuli de sécrétion et de démontrer la sécrétion de liquide 7, tandis que il a été démontré cholangiocytes isolés à développer des structures canalaire in vitro 1,2. Limites de ces méthodes comprennent la nécessité d&#39;une expertise technique spécialisée et un équipement spécial pour la perfusion du foie avec des enzymes digestives. En outre, il existe un risque de contamination par des cellules mésenchymateuses 3. Procédé pour isoler extrahépatiques cholangiocytes des canaux biliaires, en particulier de voies biliaires extra-hépatiques néonatales, n&#39;a pas été précédemment décrit. Le présent document décrit une technique simplifiée pour isoler un nouveau-nés ainsi que des cellules des canaux biliaires extra-hépatiques adultes à des niveaux élevés de pureté. Cette technique facilitera l&#39;étude des différences entre cholangiocytes et la recherche sur les mécanismes des maladies comme atrésie des voies biliaires qui impliquent les conduits extra-hépatiques et extra-hépatiques intra.

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="942">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Name of Material/Equipment</td>
			<td style="width:323px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DMEM/F12 (1:1)</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11320-033</td>
			<td>500ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">FBS</td>
			<td style="width:323px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>25 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM with non-essential amino acids</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11140-019</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Insulin-transferrin-selenium</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">51300-044</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Na Pyruvate</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-000-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Chemically-defined lipid concentrate</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11905-031</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Penicillin-Streptomycin</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">30-002-CI</td>
			<td>5ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Gentamicin</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15750-060</td>
			<td>0.2ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethanolamine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">E9508-100ml</td>
			<td>0.13ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM vitamin solution</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11120-052</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Soybean trypsin inhibitor</td>
			<td style="width:323px;">Biowhittaker</td>
			<td style="width:119px;">17-605E</td>
			<td>5ml, used in BEC media. Solvent is PBS, mix to 5mg/ml stock concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">L-glutamine</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-005-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Bovine pituitary extract</td>
			<td style="width:323px;">Gemini</td>
			<td style="width:119px;">500-102</td>
			<td>1.1ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dexamethasone</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">D4902</td>
			<td>0.5ml, used in BEC media, Stock conc 393ug/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">3 3&#39;,5-triiodo-L-thyronine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">T6397</td>
			<td>0.5ml, used in BEC media, Stock conc 3.4 mg/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Epidermal growth factor</td>
			<td style="width:323px;">millipore</td>
			<td style="width:119px;">01-101</td>
			<td>0.5ml, used in BEC media, 25ug/ml dilute with DMEM F12+1%BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Forskolin</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">F6886</td>
			<td>5ml, used in BEC media, use at stock concentration of 0.411mg/ml and dilute with DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fungizone</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15290-018</td>
			<td>1ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Rat-tail collagen</td>
			<td style="width:323px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354236</td>
			<td>variable depending on concentration of collagen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">PBS 10X</td>
			<td style="width:323px;">USB Corporation</td>
			<td align="right" style="width:119px;">75889</td>
			<td>use at 10x, sterlie, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">dH2O</td>
			<td style="width:323px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">NaOH 10N</td>
			<td style="width:323px;">Fischer Scientific</td>
			<td style="width:119px;">ss255-1</td>
			<td>Dilute to 1N, sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">collagenase type XI from Clostridium histolyticum</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">C7657</td>
			<td>dilute in DMEM and sterile filter before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (1x) 0.25%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">25200-056</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (10x) 0.5%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15400-054</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;"></td>
			<td style="width:323px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dissecting microscope</td>
			<td style="width:323px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Light source (fiberoptic illuminator)</td>
			<td style="width:323px;">Schott-Fostec</td>
			<td style="width:119px;">Ace EKE LR 92240</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">12.5 cm straight iris scissors</td>
			<td style="width:323px;">Kent Scientific</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">6&#34; non-serrated curved forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">4&#34; serrated stainless forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
	</tbody>
</table>

isolation of neonatal extrahepatic cholangiocytes

Les canaux biliaires intra et extra-hépatiques du foie sont distincts de développement, et peuvent être affectés différemment par certaines maladies. Toutefois, les différences entre cholangiocytes intra et extra-hépatiques, et entre les cellules néonatales et adultes, ne sont pas bien comprises. Méthodes pour l&#39;isolement de cholangiocytes de canaux biliaires intra-hépatiques sont bien établis 1-4. Isolement de cellules canalaires extra-hépatiques, en particulier du nouveau-né, n&#39;a pas encore été décrit, bien que ce serait d&#39;une grande utilité dans la compréhension des différences entre les populations de cholangiocytes distinctes et dans l&#39;étude des maladies telles que l&#39;atrésie biliaire qui semblent cibler les canaux extra-hépatiques. Décrit ici est une technique optimisée pour isoler les cellules des voies biliaires extra-hépatiques deux nouveau-nés et de la souris adulte. Cette technique donne une population de cellules avec une contamination minimale pur à partir de cellules mésenchymateuses telles que les fibroblastes. Cette méthod est basé sur l&#39;enlèvement des canaux extra-hépatiques et de la vésicule biliaire, suivie par une dissection minutieuse et gratter pour enlever les couches de graisse et des fibroblastes. Les structures sont noyées dans des couches épaisses de collagène et cultivées pendant 3 semaines environ pour permettre excroissance de cholangiocytes en monocouches, qui peuvent ensuite être traitées à la trypsine et étalées re pour une utilisation expérimentale.

L&#39;utilisation de ce protocole les résultats dans l&#39;isolement d&#39;une population de nouveau-nés cholangiocytes extra-hépatiques de souris avec une excellente pureté, tel que démontré par K19 immunofluorescence (Figures 2A et 2B); nous obtenons des résultats similaires à isoler des cellules de souris adultes. Nous avons observé que cela prend 3 semaines pour les cellules de canaux biliaires fraîchement isolées pour former des monocouches sur des gels de collagène épais. Les cholangi...

Watch this Scientific Journal Video about Isolation of Neonatal Extrahepatic Cholangiocytes at JoVE.com

Isolation of Neonatal Extrahepatic Cholangiocytes

Une technique pour isoler cholangiocytes des voies biliaires extra-hépatiques de souris néonatale est décrite. Les conduits sont méticuleusement disséqués, puis les cellules sont isolées par l&#39;excroissance dans des gels de collagène épaisses. Cette méthode fournit un outil utile pour étudier extrahépatique bile développement conduit et la pathologie.

Isolation des néonatale extrahépatique cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

isolation-of-neonatal-extrahepatic-cholangiocytes

Research

JoVE Journal

Biology

10.2K vues.  The Children's Hospital of Philadelphia.  Une technique pour isoler cholangiocytes des voies biliaires extra-hépatiques de souris néonatale est décrite. Les conduits sont méticuleusement disséqués, puis les cellules sont isolées par l'excroissance dans des gels de collagène épaisses. Cette méthode fournit un outil utile pour étudier extrahépatique bile développement conduit et la pathologie. 

Vidéo: Isolation of Neonatal Extrahepatic Cholangiocytes

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Isolement des neutrophiles Protocole

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Recherche

Biologie

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

L&#39;isolement des cellules endothéliales valvulaires

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolement et culture de cellules endothéliales pulmonaires des souris néonatales

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

Isolement des Drosophila melanogaster Testicules

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

Isolement et étude biophysique des cuticules de fruits

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolement et culture de cardiomyocytes de souris néonatales

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolement des cellules endothéliales embryonnaires ventriculaires

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolement de l&#39;artère pulmonaire cellules musculaires lisses de souris néonatales

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolement des cellules endothéliales murines Valve

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Isolation des néonatale extrahépatique cholangiocytes

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