Gli autori sono grati per la Patologia Molecolare e Imaging Core del UPenn NIDDK Centro Studi molecolari in Digestiva e Malattie del Fegato (P30 DK50306) per l&#39;assistenza con l&#39;imaging. Questo lavoro è stato sostenuto da sovvenzioni dal National Institutes of Health (R01 DK-092111) e dalla Fred e Suzanne Biesecker Pediatric Liver Center (a RGW) e da una borsa di studio dalla Liver Disease Research and Education Network infanzia (SK).

L&#39;intera procedura è effettuata a temperatura ambiente a meno che diversamente specificato. Tutti i lavori degli animali deve essere effettuata in condizioni umane nell&#39;ambito di un protocollo approvato dalla Institutional Animal Care ed uso comitato locale (IACUC). 1. Preparazione di apparecchiature e soluzioni <ol><li> Impostare la tabella chirurgica del mouse in prossimità della cappa coltura tissutale e incubatore (Figura 1A). Nota: Attrezzatura richiesta include un microscopio da dissezione, una fonte di luce, lungo 12,5 centimetri forbici iris diritte, pinze dentellate non sei pollici curvo con punte sottili, e 4 pollici pinza dentata con punte curve (Figura 1B). </li><li> Posizionare strumenti sterilizzati in un recipiente di vetro di 70% di alcol sul tavolo operatorio. </li><li> Riempire tre sterili 60 millimetri piastre di Petri con i media isolamento sterile (Tabella 1); collocare due sul tavolo operatorio e uno in cappuccio, tutti su ghiaccio. </li><li> Posizionare coda di topo collagene,sterile 10x tampone fosfato salino (PBS), acqua sterile, sterile 1 N NaOH e biliare cellule epiteliali (BEC) media (Tabella 1) nel cofano coltura di tessuti. Nota: Il collagene deve essere sul ghiaccio. </li></ol> 2. Isolamento Bile Duct e cultura <ol><li> Euthanize topi neonati tra 0-3 giorni di vita, in caso di esposizione al biossido di carbonio fino inconscia seguita da dislocazione cervicale, per orientamenti IACUC (Figura 1C). Nota: topi più anziani possono essere utilizzati a seconda delle specifiche esigenze sperimentali. </li><li> Posizionare animale in posizione supina e fare una piccola incisione orizzontale che attraversa la linea mediana della regione del bacino. Estendere questa incisione laterale e superiore entrambi i lati del ventre, formando un lembo a forma di U. Girare lembo fino a esporre gli organi addominali. Una volta che l&#39;addome è aperto, spostare con cautela l&#39;intestino dalla cavità addominale verso il lato destro dell&#39;animale per una migliore visualizzazione del dotto epatico e biliare comune. Nota: può essere necessario estendere le incisioni laterali nella gabbia toracica avere una migliore identificazione del fegato. </li><li> Utilizzando il microscopio da dissezione, identificare il dotto biliare comune, fegato e intestino. Identificare il dotto biliare comune prima (Figura 1D), con cautela, in quanto le strutture sono molto delicati. Con le pinze dentellate seghettati e non, meticolosamente puliti e far fuori il tessuto connettivo incluso il grasso che circonda la bile. Utilizzare una pinza per tenere il condotto e l&#39;altro da pulire. </li><li> Utilizzare una tecnica simile per pulire la cistifellea. </li><li> Posizionare i condotti puliti e cistifellea nel primo piatto di terreni di isolamento sul ghiaccio. Massaggiare delicatamente le strutture con le pinze non dentati, mentre nei media per eliminare ulteriormente il tessuto connettivo e togliere la bile dalla cistifellea. </li><li> Trasferire condotti e cistifellea al secondo piatto di terreni di isolamento. </li><li> Dopo l&#39;isolamento dei condotti e colecisti da 5 animali, trasferire ilpezzi al terzo piatto Petri, nella cappa. Nota: Se le cellule devono essere isolato da più di 5 animali, fare in batch, utilizzando soluzioni fresche per ogni gruppo di 5 animali. </li><li> Preparare gel di collagene di spessore nella cappa coltura tissutale: Nota: ogni piatto formato può essere utilizzato. Un piatto di coltura 60 mm Con un volume finale di 3 ml di collagene per ogni gruppo di 5 neonati funziona bene. Questo deve essere preparato fresco al momento di incorporare condotti appena isolate. <ol><li> Posizionare ratto collagene coda, sterile 10x tampone fosfato (10x PBS), dH 2 O sterile e sterile 1 N NaOH su ghiaccio. </li><li> Calcolare i volumi di dH 2 0, 10x PBS, NaOH, e collagene necessario per gel con una concentrazione finale di 2 collagene mg / ml in PBS 1x, utilizzando un volume di 1 N NaOH pari a 0.023 volte il volume di collagene memo. Nota: Se si usa fonte alternativa di collagene, seguire le istruzioni del produttore per la neutralizzazione e la formazione di gel. </li><li> Mescolare dH 2 0, 10x PBS e 1 N NaOH, quindi aggiungere collagene e pipetta bene per assicurare una miscelazione uniforme. </li></ol></li><li> Una soluzione di collagene è addensata ad un gel come consistenza a temperatura ambiente, immediatamente incorporare i condotti del collagene. </li><li> Porre in incubatore a 37 ° C, 5% CO 2 per 30 minuti per consentire al collagene a solidificare. </li><li> Una volta che il gel si è solidificato (cambiamenti di colore dal chiaro al bianco), sovrapposizione con 3,5 ml di mezzi di BEC (Tabella 1) e incubare a 37 ° C, 5% di CO 2. </li><li> Rimuovere i supporti BEC 3 volte a settimana e sostituirlo con 3,5 ml di mezzi freschi. </li></ol> 3. Isolanti primari colangiociti da Thick Collagene Gel Nota: entro 3 settimane, fogli di colangiociti (cellule con grandi nuclei che si estende direttamente dai condotti incorporati) saranno visibili nel collagene di spessore. Se sono presenti un gran numero di fibroblasti nel piatto, deve essere eliminato. Se alcune regioni del piatto hannocrescita dei fibroblasti, questi può essere tagliato con un bisturi e rimosso prima di dividere i colangiociti. <ol><li> Preparare gel di collagene sottili: <ol><li> Diluire collagene ad una concentrazione di 1 mg / ml con PBS sterile. Nota: utilizzare 1 ml per piastra mm 100; regolare di conseguenza per gli altri piatti di dimensioni. </li><li> Diffondere la soluzione di collagene uniformemente con una spatola cellulare (piatti di grandi dimensioni) o la punta della pipetta (piccoli piatti), poi lasciare a temperatura ambiente per 10 min. </li><li> Coprire piastra con DMEM/F12 e incubare a 37 ° C, 5% CO 2 per 10 min. </li><li> Rimuovere la piastra da incubatore e lavare con PBS 1x. Aspirare immediatamente la 1x PBS. </li><li> Coat con un secondo strato di collagene, diffondendo ruotando la piastra o immissione piastra su un agitatore. Incubare a 37 ° C, 5% CO 2 per 10 min. </li><li> Dopo collagene è solidificato, piastra di copertura con DMEM/F12 e incubare a 37 ° C, 5% CO 2 per 30 min. </li></ol></li><li> Dividere le celle da thick gel di collagene: <ol><li> Con la coltura cellulare spatola o un raschietto cellula, raschiare gel di collagene delicatamente la piastra in modo che galleggi. </li><li> Preparare la soluzione collagenasi diluendo tipo XI collagenasi (vedi elenco materiali) a 10 mg / ml con DMEM/F12. Mescolare bene e filtro sterile. </li><li> Aggiungere 1 ml di soluzione di collagenasi (sopra) ogni 60 mm senza coperchio di cellule in collagene di spessore. Non rimuovere il supporto BEC (che dovrebbe essere di 3 ml). Incubare piatto per 30 min a 37 ° C, 5% CO 2. </li><li> Rimuovere la soluzione contenente le cellule, e metterli in tubo da 15 ml. </li><li> Spin down a 2.200 xg per 5 min. Eliminare il surnatante e sospendere nuovamente pellet in volume analogo di DMEM/F12. </li><li> Spin nuovamente la soluzione a 2.200 xg per 5 min. Rimuovere il surnatante. </li><li> Risospendere pellet in 3 ml di 0,5% tripsina, e incubare a 37 ° C, 5% CO 2 per 5 min. Dopo l&#39;incubazione, pipetta la soluzione su e giù per rompere i fogli di colangiociti. Aggiungere 5 ml di BEC supporti unsoluzione centrifuga nd a 1.500 xg per 5 min. </li><li> Rimuovere il surnatante, risospendere pellet e in DMEM/F12, poi girare soluzione a 1.500 xg per 5 min. </li><li> Rimuovere il surnatante e sospendere nuovamente pellet nei media BEC (Tabella 1). Cellule piastra su fatte in precedenza gel di collagene sottili. </li></ol></li></ol> 4. Passage e stoccaggio delle cellule Nota: celle su gel di collagene sottili possono essere suddivise come necessario. <ol><li> Lavare le piastre con PBS sterile prima dell&#39;incubazione a 0,25% tripsina a 37 ° C per 10-15 min. </li><li> Neutralizzare con un volume uguale di mezzi BEC e pellet a 1500 xg per 5 min. Risciacquare pellet con DMEM/F12, poi pellet a 1500 xg per 5 min. </li><li> Risospendere le cellule in mezzi BEC per la distribuzione di collagene rivestito di coltura cellulare piatti. Nota: In alternativa, le cellule possono essere risospese in supporti di memorizzazione e conservati in un congelatore -80 ° C o in azoto liquido (Tabella 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>

Gli autori dichiarano di non avere interessi in competizione.

Qui descritto è una tecnica per isolare colangiociti puri dai dotti biliari extraepatici di topi di topi di tutte le età, compresi i neonati. La tecnica offre il vantaggio che colangiociti extraepatici possono essere studiate separatamente da colangiociti intraepatiche, e può facilitare studi per individuare le differenze principali tra queste popolazioni di cellule. Abbiamo recentemente pubblicato una dimostrazione studio diminuito ciglia in colangiociti extraepatiche isolato con questo metodo e con infezione da rot...

L&#39;origine e lo sviluppo dei colangiociti intra ed extra sono nettamente diverse. Il fegato si sviluppa da un diverticolo della ventrale foregut endoderma 5. La regione caudale del diverticolo forma dell&#39;albero biliare extraepatico, mentre la regione craniale genera l&#39;albero biliare intraepatico 5. Colangiociti dei dotti intraepatici sono derivati ​​da cellule progenitrici intorno alla piastra duttale in peri regioni portale 6. Queste cellule hanno la capacità di differenziarsi in epatociti o colangiociti sia 6. Ciò ha importanti implicazioni cliniche dato che cholangiopathies possono rivolgono specificamente una categoria di colangiociti. Ad esempio, l&#39;atresia biliare colpisce inizialmente i dotti extraepatici del neonato, mentre la sindrome di Alagille colpisce dotti intraepatici. Ci sono diverse descrizioni di isolamento delle colangiociti intraepatici e unità del dotto biliare da topi e ratti. Investigators hanno isolato cellule singole dall&#39;isolamento condotto e la successiva escrescenza, o dalla digestione fegato e anticorpo abbattere di colangiociti esprimono specifici marcatori di superficie delle cellule 1-4. Unità del dotto biliare funzionali e polarizzati sono stati isolati mediante digestione fegato e dimensioni filtrazione 7. Unità dotto biliare sono in grado di rispondere alle secretoria stimoli e dimostrare la secrezione di fluidi 7, mentre colangiociti isolati hanno dimostrato di sviluppare strutture duttulare in vitro 1,2. Limitazioni di questi metodi includono la necessità di competenze tecnico specializzato e attrezzature speciali per la perfusione del fegato con enzimi digestivi. Inoltre, vi è il rischio di contaminazione con cellule mesenchimali 3. Un metodo per isolare colangiociti dei dotti biliari extraepatiche, in particolare da dotti biliari extraepatici neonatale, non è stato descritto in precedenza. Il documento illustra una tecnica semplificata per isolare neonatale uns così come le cellule adulte dotti biliari extraepatici ad alti livelli di purezza. Questa tecnica faciliterà lo studio delle differenze tra colangiociti e ricerche sui meccanismi di malattie come l&#39;atresia biliare che coinvolgono i dotti extraepatici intra ed extraepatiche.

<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

I dotti biliari intra ed extraepatici del fegato sono evolutivamente distinti, e possono essere differenzialmente influenzati da certe malattie. Tuttavia, le differenze tra colangiociti intra ed extra, e tra le cellule neonatali e adulte, non sono ben compresi. Metodi per l&#39;isolamento di colangiociti da dotti biliari intraepatici sono ben stabiliti 1-4. Isolamento di cellule duttali extraepatici, specialmente dal neonato, non è ancora stato descritto, anche se questo sarebbe di grande beneficio per comprendere le differenze fra le popolazioni colangiociti distinte e nello studio malattie come atresia biliare che sembrano indirizzare i dotti extraepatici. Qui descritto è una tecnica ottimizzata per isolare le cellule dei dotti biliari extraepatici sia neonatali e topo adulto. Questa tecnica produce una popolazione pura di cellule con contaminazioni minimo da cellule mesenchimali come fibroblasti. Questa metodologiad è basato sulla rimozione dei dotti extraepatici e cistifellea, seguita da dissezione meticolosa e raschiatura per rimuovere strati di grasso e fibroblasti. Le strutture sono incorporati in spessi strati di collagene e coltivate per circa 3 settimane per consentire conseguenza di colangiociti in monostrati, che possono poi essere tripsinizzato e ri placcato per uso sperimentale.

L&#39;uso di questo protocollo risultati in isolamento di una popolazione di neonatali di topo colangiociti extraepatici con ottima purezza, come dimostrato da K19 immunofluorescenza (Figure 2A e 2B); otteniamo risultati simili isolano cellule di topi adulti. Abbiamo osservato che ci vogliono 3 settimane per cellule da dotti biliari appena isolate per formare monostrati su gel di collagene di spessore. I colangiociti crescono in modo lineare durante il collagene spessa, fogli di formazione di cellule da...

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

Isolation of Neonatal Extrahepatic Cholangiocytes

Una tecnica per isolare colangiociti dai dotti biliari extraepatici di topi neonati è descritto. I condotti sono meticolosamente sezionato, e poi le cellule sono isolate da escrescenza in gel di collagene di spessore. Questo metodo fornisce un utile strumento per lo studio dello sviluppo del dotto biliare extraepatico e patologia.

Isolamento dei neonatale extraepatico colangiociti

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 Views.  The Children's Hospital of Philadelphia.  Una tecnica per isolare colangiociti dai dotti biliari extraepatici di topi neonati è descritto. I condotti sono meticolosamente sezionato, e poi le cellule sono isolate da escrescenza in gel di collagene di spessore. Questo metodo fornisce un utile strumento per lo studio dello sviluppo del dotto biliare extraepatico e patologia. 

Video: 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.

Neutrofili isolamento Protocollo

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

Ricerca

Biologia

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.

Isolamento di cellule endoteliali valvolare

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.

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

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.

Isolamento di Drosophila melanogaster Testicoli

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.

Isolamento e studio della biofisica cuticole di frutta

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.

Isolamento e Cultura della neonatali mouse Cardiomyocytes

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.

Isolamento della embrionali ventricolari cellule endoteliali

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.

Isolamento di polmonare cellule muscolari lisce di topi neonati

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.

Isolamento di cellule murine endoteliali 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.