Ringraziamo lo staff di AniCan (CrCl, Lione), per la loro assistenza tecnica con la cura degli animali. Questo lavoro è stato sostenuto da l&#39;Institut National de la Santé et de la Recherche Médicale (INSERM Avenir Program), la Ligue Nationale contre le Cancer, dall&#39;Associazione pour la Recherche sur le Cancer, dall&#39;Institut National du Cancer, e da borse di studio da la Ligue Nationale Contre le Cancer (JG), presso l&#39;Institut National du Cancer (JG), dal Ministère de l&#39;Enseignement Supérieur et de la Recherche di Francia (RMP e DFV) e dalla Association pour la Recherche sur le Cancer ( DFV).

Tutte le procedure sono state approvate da un comitato etico sotto regolamentazione dei pubblici poteri (&quot;Comité d&#39;Evaluation Commun Au Centre Léon Bérard, à l&#39;Animalerie de transito de l&#39;ENS, au PBES et au laboratoire P4&quot; (CECCAPP)). I topi sono stati mantenuti in una specifica struttura di animale privo di patogeni presso la &quot;Piattaforma AniCan, Centro Léon Bérard&quot; (Lione, Francia) e trattati in conformità con le linee guida istituzionali. Una rappresentazione schematica del procedimento è mostrato in Figura 1. 1. Pancreas Dissection e dilacerazione (giorno 0) Una molto rapida dissezione è critica per una resa ottimale di estrazione e per assicurare una buona vitalità delle cellule in coltura. Per ridurre il tempo necessario per l&#39;isolamento pancreas, tutti gli strumenti e le attrezzature devono essere pronti prima della eutanasia mouse. <ol><li><html> Euthanize topo da CO 2 asfissia o dislocazione cervicale. </li></ol> Da questo punto, tutte le procedure devono essere eseguite sotto un&#39;atmosfera sterile (microbiologica, livello II) con attrezzature dissezione sterile. <ol start="2"><li> Fissare il mouse e spruzzare l&#39;addome del mouse con il 70% di etanolo. Con le forbici dissezione e pinze, fare un&#39;incisione a forma di V nella zona genitale e continuare fino al diaframma di aprire completamente la cavità addominale. </li><li> Posizionare i lobi del fegato contro il diaframma, che devono rimanere lì, se la cavità del corpo è aperta abbastanza lontano. Tirare l&#39;intestino e il colon fuori della cavità addominale alla vostra sinistra, e trovare il retto. Con un paio di pinze curve e forbici dissezione, afferrare e la sezione del retto. </li><li> Con la stessa coppia di pinze, accuratamente srotolare completamente l&#39;intestino dal retto allo stomaco nell&#39;intestino tirando sulla sinistra. </li></ol>ent &quot;&gt; In questo passaggio, il pancreas può essere distinto da una piccola striscia tra lo stomaco e l&#39;inizio del viscere. sue legature con la milza rimangono intatti. <ol start="5"><li> Utilizzando Noyes forbici e un paio di pinze, tagliare con cautela il pancreas lungo l&#39;intestino e liberare con la milza dal resto del tratto digestivo. </li><li> Afferrare la sezione di milza e il pancreas collegato ad esso (Figura 2). </li></ol> In questo passaggio, assicurarsi che nessun tessuto grasso mesenterico e / o di altri tessuti adiacenti (milza, intestino, ecc) potrebbero essere raccolti con il pancreas, per evitare la contaminazione cellulare. Per il resto della procedura, tutti i buffer devono essere preparate senza calcio ione Ca 2 + chelanti per evitare la completa dissociazione del tessuto pancreatico esocrino nelle cellule acinose singoli. <ol start="7"><li> Sciacquare il pancreas due volte in soluzione salina bilanciata di Hank (HBSS) 1x. </li></ol> In questa fase, come tessuto grasso galleggia contraria pancreas che affonderà, è facilmente possibile visualizzare e rimuovere rapidamente il contaminante tessuto adiposo bianco ancora attaccato al pancreas. Se il pancreas deve essere trasportato alla struttura di coltura cellulare, deve essere tenuta in ghiaccio in HBSS 1x. <ol start="8"><li> Trasferire il pancreas in una piastra di Petri sterile contenente 5 ml di HBSS 1x. Utilizzando Noyes forbici e un bisturi, tagliare il pancreas in piccoli pezzi di 1 a 3 mm 3 (Figura 3A). </li></ol> 2. Dissociazioni enzimatica e meccanica di pancreas (giorno 0) <ol><li> Trasferirli in un tubo da 50 ml in polipropilene sterile. </li><li> Centrifugare per 2 minuti a 450 xg e 4 ° C. Aspirare e scartare il surnatante per rimuovere frammenti di cellule e le cellule del sangue. </li><li> Aggiungere 10 ml di soluzione di collagenasi IA (HBSS 1x contenente HEPES 10 mm, 200 U / ml di collagenaSE IA, e 0,25 mg / ml di inibitore della tripsina) alle sezioni pancreas. Usando una pipetta sierologica 25 ml, trasferirli su un 2 tarato da 25 cm. Incubare per 20-30 minuti a 37 ° C. Durante questo periodo (ogni 5 minuti), eseguire una dissociazione meccanica energicamente spostando avanti e indietro i frammenti di pancreas di circa dieci volte, in pipette sterili di dimensioni decrescenti (25, 10 e 5 ml pipette sierologiche). </li></ol> In questa fase, è essenziale monitorare frequentemente l&#39;entità della dissociazione enzimatica di sezioni pancreatiche. <ol start="4"><li> Quando il tessuto pancreatico sembra essere ben dissociato (secondo la scomparsa di frammenti pancreatici e alla maggiore torbidità della soluzione) (Figura 3B), arrestare la reazione enzimatica aggiungendo 10 ml di soluzione di lavaggio tamponata freddo (1x HBSS contenente 5 % siero fetale bovino (FBS) e 10 mM HEPES). </li><li> Trasferire in un tubo da 50 ml in polipropilene sterile e centrifuge per 2 min a 450 xg e 4 ° C. Con attenzione aspirare e scartare il surnatante per rimuovere la soluzione di collagenasi IA. </li><li> Risospendere il pellet e lavare con 10 ml di soluzione di lavaggio tamponata. Centrifugare per 3 minuti a 450 xg e 4 ° C. Con attenzione aspirare e scartare il surnatante. Ripetere questa operazione per altre due volte. </li></ol> 3. Filtrazione e la semina dei Dispersi Acini (giorno 0) <ol><li> Risospendere il pellet cellulare in 7 ml di mezzo di Waymouth contenente 2,5% FBS, 1% penicillina-streptomicina miscela (PS), 0,25 mg / ml di inibitore della tripsina, e 25 ng / ml di fattore di crescita epidermico umano ricombinante (EGF). </li><li> Filtrate il composto cella, consentendo di passare attraverso un 100 micron filtro di trattenere i frammenti non digeriti (condotti, vasi sanguigni, e le isole di Langerhans). Strutture acinose pancreatiche (acino di 10-15 cellule) passano attraverso. </li><li> Lavare il filtro con 6 ml di terreno contenente FBS di Waymouth, PS, tripsina inhibitor e EGF. </li></ol> Dopo questo passo, le cellule devono essere trattati con molta attenzione, per evitare qualsiasi acini dissociazione. <ol start="4"><li> Seme acini isolato in un piatto di coltura a 6 pozzetti (2 ml per pozzetto) (Figura 3C). Li coltura a 37 ° C sotto 5% (v / v) di CO 2 nell&#39;atmosfera. </li></ol> Dopo questo passo, le cellule acinose sono coltivate in sospensione. 4. Acinose Cell Culture (Giorno 1 a 10) <ol><li> Ventiquattro ore dopo, trasferire il acini (in sospensione) in un nuovo piatto di coltura a 6 pozzetti, per eliminare le cellule contaminanti ei residui cellulari che hanno aderito overnight (Figura 4). </li></ol> Se la coltura cellulare deve essere ampliato per diversi giorni o se le condizioni sperimentali richiedono cellule cresciute in monostrato, si raccomanda di trasferimento e sementi acini sulla matrice scaffold. <ol start="2"><li> Il giorno prima di vedereding sul supporto matrice (giorno 0), cappotto un piatto di coltura da 6 pozzetti con collagene di tipo I (5 pg / cm 2). Aggiungere 1 ml di soluzione di collagene di tipo I (50 ug / ml in 0,02 M di acido acetico, 0,2 micron filtrata) ad ogni pozzetto e permettono di adsorbire passivamente su plastica, durante 1 ora a 37 ° C (o per una notte a 4 ° C ). </li><li> Aspirare la soluzione di collagene di tipo I e sciacquare il rivestimento ben due volte con PBS 1x. </li><li> Lasciare che il rivestimento oltre ad asciugare (sotto una cappa di sicurezza microbiologica) almeno 12 ore prima dell&#39;uso. </li><li> Trasferire l&#39;isolato primario acini (ottenuto nella Fase 4.1) nel collagene tipo I-rivestito piatto di coltura a 6 pozzetti e cultura nelle stesse condizioni come precedentemente descritto (a 37 ° C sotto 5% (v / v) di CO 2 nell&#39;atmosfera) . Le cellule aderiscono al substrato di collagene di tipo I per 2 giorni. </li><li> Il giorno 3, modificare il terreno di coltura per eliminare le cellule non vitali, che non hanno aderito. Con il tempo in coltura, le cellule saranno progressivamente spleggere sul supporto contenente collagene. Cambia il terreno di coltura ogni 3 giorni (Figura 5). </li></ol> Le cellule acinose isolate ottenute possono essere contate, dopo una completa dissociazione meccanica utilizzando una camera di conteggio delle cellule Thoma. Si noti che le cellule acinose isolate non possono essere mantenute in coltura in seguito. La qualità della cultura acinose ottenuto può essere controllata controllando l&#39;espressione di marcatori specifici acinose quali Trypsinogen, Pancreas Transcription Factor 1 subunità alfa, o Carboxypeptidase A1 (mediante immunocitochimica o esperimenti di immunofluorescenza).

<ol>
	<li>Lardon, J., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaplasia+in+the+pancreas.">Metaplasia in the pancreas.</a> Differentiation. 73, 278-286 (2005).</li><li>Sphyris, N., Logsdon, C. D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+retention+of+zymogen+granules+in+cultured+murine+pancreatic+acinar+cells+and+induction+of+acinar-ductal+transdifferentiation+in+vitro.">Improved retention of zymogen granules in cultured murine pancreatic acinar cells and induction of acinar-ductal transdifferentiation in vitro.</a> Pancreas. 30, 148-157 (2005).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characterization+of+isolated+pancreatic+exocrine+cells.">Structural and functional characterization of isolated pancreatic exocrine cells.</a> Proc. Natl. Acad. Sci. U.S.A. 69, 3028-3032 (1972).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+dispersed+pancreatic+exocrine+cells.+I.+Dissociation+technique+and+morphologic+characteristics+of+separated+cells.">Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells.</a> J. Cell. Biol. 63, 1037-1056 (1974).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+dispersed+pancreatic+exocrine+cells.+II.+Functional+characteristics+of+separated+cells.">Studies on dispersed pancreatic exocrine cells. II. Functional characteristics of separated cells.</a> J. Cell. Biol. 63, 1057-1073 (1974).</li><li>Schultz, G. S., 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=Guinea+pig+pancreatic+acini+prepared+with+purified+collagenase.">Guinea pig pancreatic acini prepared with purified collagenase.</a> Exp. Cell Res. 130, 49-62 (1980).</li><li>Williams, J. A., Korc, M., Dormer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+secretagogues+on+a+new+preparation+of+functionally+intact,+isolated+pancreatic+acini.">Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.</a> Am. J. Physiol. 235, 517-524 (1978).</li><li>Logsdon, C. D., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+binding+and+biologic+effects+on+mouse+pancreatic+acini.">Epidermal growth factor binding and biologic effects on mouse pancreatic acini.</a> Gastroenterology. 85, 339-345 (1983).</li><li>Han, B., Logsdon, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholecystokinin+induction+of+mob-1+chemokine+expression+in+pancreatic+acinar+cells+requires+NF-kappaB+activation.">Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-kappaB activation.</a> Am. J. Physiol. 277, 74-82 (1999).</li><li>Ji, B., Kopin, A. S., Logsdon, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+differences+between+rat+and+mouse+CCKA+receptors+determine+the+divergent+acinar+cell+response+to+the+cholecystokinin+analog+JMV-180.">Species differences between rat and mouse CCKA receptors determine the divergent acinar cell response to the cholecystokinin analog JMV-180.</a> J. Biol. Chem. 275, 19115-19120 (2000).</li><li>Ji, K. A., Yang, M. S., Jou, I., Shong, M. H., Joe, E. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+acinar+cells+in+monolayer+culture:+direct+trophic+effects+of+caerulein+in+vitro.">Pancreatic acinar cells in monolayer culture: direct trophic effects of caerulein in vitro.</a> Am. J. Physiol. 250, 440-447 (1986).</li><li>Stanger, B. Z., Dor, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+cellular+origins+of+pancreatic+cancer.">Dissecting the cellular origins of pancreatic cancer.</a> Cell Cycle. 5, 43-46 (2006).</li><li>Vincent, D. F., 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=Tif1gamma+suppresses+murine+pancreatic+tumoral+transformation+by+a+smad4-independent+pathway.">Tif1gamma suppresses murine pancreatic tumoral transformation by a smad4-independent pathway.</a> Am. J. Pathol. 180, 2214-2221 (2012).</li><li>Vincent, D. F., 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=Inactivation+of+TIF1gamma+cooperates+with+Kras+to+induce+cystic+tumors+of+the+pancreas.">Inactivation of TIF1gamma cooperates with Kras to induce cystic tumors of the pancreas.</a> PLoS Genet. 5, e1000575 (2009).</li><li>Dorrell, C., 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=Isolation+of+mouse+pancreatic+alpha,+beta,+duct+and+acinar+populations+with+cell+surface+markers.">Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.</a> Mol. Cell Endocrinol. 339, 144-150 (2011).</li><li>Case, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+intracellular+transport+and+discharge+of+exportable+proteins+in+the+pancreatic+acinar+cell+and+other+cells.">Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cell and other cells.</a> Biol. Rev. Camb. Philos. Soc. 53, 211-354 (1978).</li><li>Blauer, M., Nordback, I., Sand, J., Laukkarinen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+explant+outgrowth+culture+model+for+mouse+pancreatic+acinar+cells+with+long-term+maintenance+of+secretory+phenotype.">A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype.</a> Eur. J. Cell. Biol. 90, 1052-1060 (2011).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

In questo protocollo, si descrive una procedura per isolare le cellule acinose pancreatiche. Questo metodo rende possibile isolare più di 20 x 10 6 cellule acinose per animale in meno di 1 ora. Grazie alla sua attuazione rapida e semplice (ben 10 pancreas possono essere trattati in modo indipendente per ogni esperimento in parallelo), questo protocollo viene visualizzato come un buon compromesso tra i metodi di isolamento esistenti 3-5,9-12,17. Passaggi critici / r...

Un problema riscontrato frequentemente per laboratori di ricerca lavorano su tessuto pancreatico esocrino è la difficoltà di coltivare cellule acinose in vitro per un periodo di tempo sufficientemente lungo da consentire un esperimento a lungo termine. Un fattore impedendo lo sviluppo di tali sistemi di coltura è la sensibilità intrinseca del tessuto pancreatico di manipolazione sperimentale a causa dell&#39;elevato contenuto di glicolitiche, proteolitica e lipolitica enzimi, che letteralmente digeriscono il tessuto pancreatico quando vengono rilasciate durante l&#39;isolamento delle cellule pancreatiche. Un secondo fattore è la notevole plasticità in vitro di cellule acinose, che tendono a perdere le loro caratteristiche secretorie e transdifferenziarsi ad altre cellule mature, quali le cellule pancreatiche duttali o cellule epatociti-simili 1. In vitro, questa plasticità cella varia con le condizioni sperimentali (come composizione mezzo di coltura) 2 </sup&gt; e introduce un grado di complessità nella progettazione di condizioni di coltura appropriati per le cellule pancreatiche esocrine 1. Diversi metodi sono stati sviluppati per l&#39;isolamento e la coltura di cellule acinose, prima della cavia pancreas 3-5. Inizialmente, tali protocolli coinvolti digestione del tessuto pancreatico con collagenasi, chimotripsina, e un cocktail proteasi, con l&#39;isolamento definitivo da vigoroso dissociazione meccanica. Le cellule pancreatiche isolate in questo modo visualizzati caratteristiche strutturali e funzionali anomale, in particolare una perdita di strutture apicali e danni significativi ai loro recettori di membrana. Cellule isolate rimaste vitali per solo 1 o 2 giorni. Preparazione di disperso acini mantiene la loro architettura intra-e intercellulare, preservando le membrane cellulari, limitando i danni ai recettori della superficie, e quindi migliorando la secrezione esocrina in risposta ai secretagoghi 6-8. Come result, questo metodo offre il grande vantaggio di estendere vitalità cellulare acinose per 7-10 giorni in vitro. Inoltre, questo metodo viene attualmente preferito isolamento delle cellule acinose 9-12 perché la manutenzione dei contatti intercellulari, tra accoppiamento cellula per giunzioni, è un determinante essenziale del pancreatica esocrina acinoso fenotipo delle cellule 13. Come dedifferentiation delle cellule acinose e la loro transdifferenziazione di cellule duttali è uno dei meccanismi proposti per la genesi di tumori del pancreas esocrino aggressivi 14, il modello disperso acini è anche un sistema adeguato per studiare plasticità pancreatica e successive meccanismi molecolari. Inoltre, in combinazione con l&#39;uso di animali geneticamente modificati 15,16 e lo sviluppo di tecniche di trasferimento genico (2 adenovirale o trasduzione lentivirale, uso di nanoparticelle, etc), questo modello in vitro cellule acinose primario puòessere molto utile per determinare come le varie disfunzioni genetiche influenzano la regolazione della differenziazione delle cellule acinose o de-differenziazione e dovrebbero fornire una migliore comprensione degli eventi molecolari responsabili della insorgenza di pancreatite, lesioni precancerose, e cambiamenti nella plasticità delle cellule. Isolamento dei dispersi acini è l&#39;approccio che usiamo nel nostro laboratorio di cultura pancreatiche cellule dell&#39;acino. Siamo qui descriviamo e discutiamo il metodo utilizzato. Esso comporta dissociazione enzimatica del tessuto pancreatico (con collagenasi batterica) accoppiato a rottura meccanica senza dissociazione delle cellule acinose. Mentre la maggior parte dei protocolli prevedono la coltura degli acini, in sospensione o su substrati plastici appositamente trattati, li cresciamo in sospensione solo brevemente (per 24 ore), la semina loro in seguito su scaffold di matrice se è richiesta la coltura cellulare prolungato. Questo protocollo permette isolamento rapido (in meno di 1 ora) di dispersi pancreatica acini, sostenibile per più di una settimana di cultura. Esso consente l&#39;isolamento di più di 20 x 10 6 cellule acinose al pancreas mouse. La sua semplicità consente di elaborare in modo indipendente fino a 10 pancreas in parallelo. Mantenendo l&#39;architettura intra-e intercellulare di acini e quindi il fenotipo di cellule acinose primarie isolate, questo modello costituisce un sistema di scelta per lo studio dei meccanismi transdifferenziazione, come tutti gli altri modelli pancreas esocrino attualmente disponibili sono derivate da tumori pancreatici visualizzare molte genetica alterazioni porta alla trasformazione cellulare.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>0.2 &mu;m filter</td>
			<td>Dutscher</td>
			<td>146560</td>
			<td></td>
		 </tr>
		 <tr>
			<td>10 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>100 &mu;m filter</td>
			<td>Beckton Dickinson</td>
			<td>352360</td>
			<td></td>
		 </tr>
		 <tr>
			<td>100 mm Petri dish</td>
			<td>Beckton Dickinson</td>
			<td>353003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1000 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1122-1830</td>
			<td></td>
		 </tr>
		 <tr>
			<td>20 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1120-1810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>200 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1120-8810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>25 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357535</td>
			<td></td>
		 </tr>
		 <tr>
			<td>5 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357543</td>
			<td></td>
		 </tr>
		 <tr>
			<td>50 ml polypropylene tube</td>
			<td>Beckton Dickinson</td>
			<td>352070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>6-well plate</td>
			<td>Beckton Dickinson</td>
			<td>353046</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetic acid 100%</td>
			<td>VWR BDH Prolabo</td>
			<td>20104.298</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagenase IA</td>
			<td>Sigma-Aldrich</td>
			<td>C2676</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Curved forceps, Dumont #7</td>
			<td>World Precision Instruments</td>
			<td>14188</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Dissecting scissors, straight</td>
			<td>World Precision Instruments</td>
			<td>14393</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Epidermal Growth Factor, human</td>
			<td>Promokine</td>
			<td>C-60180</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethanol absolute (AnalaR Normapur)</td>
			<td>VWR BDH Prolabo</td>
			<td>20821.310</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-801F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Forceps, Dumont #5</td>
			<td>World Precision Instruments</td>
			<td>14098</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Hank's Balanced Salt Solution 1x </td>
			<td>Gibco</td>
			<td>14025050</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HEPES 1 M (pH 6.98-7.30)</td>
			<td>Lonza</td>
			<td>17-737F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Incubator O2/CO2</td>
			<td>Sanyo</td>
			<td>MCO-19M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Matrigel</td>
			<td>Beckton Dickinson</td>
			<td>356234</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microbiological Safety Cabinet, level II</td>
			<td>Faster</td>
			<td>SafeFast Elite 212 S</td>
		 </tr>
		 <tr>
			<td>Noyes scissors, sharp/sharp tips, German</td>
			<td>World Precision Instruments</td>
			<td>500228-G</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Penicillin-Streptomycin mixture</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffer Saline 10x </td>
			<td>Gibco</td>
			<td>14200067</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipet-Aid</td>
			<td>Drummond Scientific Company</td>
			<td>Pipet-Aid XP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scalpel</td>
			<td>Paramount Surgimed Ltd.</td>
			<td>Disposable Scalpel Size 23</td>
		 </tr>
		 <tr>
			<td>T25 flask, 25 cm2</td>
			<td>Sigma-Aldrich</td>
			<td>Z707481</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin inhibitor, from Glycine Max</td>
			<td>Sigma-Aldrich</td>
			<td>T6522</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Type I collagen</td>
			<td>Beckton Dickinson</td>
			<td>354236</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Waymouth's medium</td>
			<td>Gibco</td>
			<td>31220-023</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation and culture of mouse primary pancreatic acinar cells

Questo protocollo permette isolamento rapido (in meno di 1 ora) di acini pancreas murino, rendendo possibile mantenerli in coltura per più di una settimana. Più di 20 x 10 6 cellule acinose possono essere ottenuti da un singolo pancreas murino. Questo protocollo offre la possibilità di elaborare separatamente ben 10 pancreases in parallelo. Perché conserva architettura acinose, questo modello è adatto per studiare la fisiologia del pancreas esocrino in vitro in contrasto con linee cellulari stabilizzate da tumori pancreatici, che visualizzano molte alterazioni genetiche conseguente perdita parziale o totale della loro differenziazione acinose.

La figura 1 schematizza la &quot;dispersa&quot; metodo acini per primario cellule acinose isolamento. Fasi critiche, che devono essere rigorosamente rispettati durante il protocollo, sono descritti nella parte discussione. Per facilitare la sua rimozione, il pancreas deve essere raccolta dall&#39;addome insieme alla milza allegato (figura 2). Entrambi gli organi devono essere tagliate a parte, e il tessuto grasso residuo che potrebbe essere ancora attaccato ...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Primary Pancreatic Acinar Cells at JoVE.com

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

In questa pubblicazione, si descrive una procedura rapida e conveniente per l&#39;isolamento e la coltura primaria cellule pancreatiche acinose del pancreas murino. Questo metodo costituisce un valido approccio per studiare la fisiologia delle cellule esocrine del pancreas freschi primarie normali / non trasformata.

Isolamento e Cultura del mouse principale acinose pancreatiche Cellule

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

isolation-and-culture-of-mouse-primary-pancreatic-acinar-cells

Research

JoVE Journal

Biology

35.4K Views.  Centre de Recherche en Cancérologie de Lyon.  In questa pubblicazione, si descrive una procedura rapida e conveniente per l'isolamento e la coltura primaria cellule pancreatiche acinose del pancreas murino. Questo metodo costituisce un valido approccio per studiare la fisiologia delle cellule esocrine del pancreas freschi primarie normali / non trasformata. 

Video: Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

Isolamento di cellule primarie trofoblasto mouse e Invasion Assay trofoblasto

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Ricerca

Biologia

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolamento e la coltura primaria di cellule epatiche di ratto

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex&#160;vivo culture will represent the in&#160;vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

Isolated pancreatic acini retain their in&#160;vivo morphology and activity and offer powerful ways for monitoring and manipulating secretion. This work demonstrates how acini can be isolated from the mouse pancreas, and how their secretory capacities can be assessed.

Valutare la capacità secretoria delle cellule pancreatiche acinari

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e colture primarie di cellule di topo aortica endoteliali

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e cultura dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Isolamento e cultura delle cellule endoteliali aortiche primarie dai suini in miniatura

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Dissezione e coltura efficienti delle cellule epiteliali del pigmento retino del topo primario

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolamento e coltura di cheratinociti orali primari dal palato del topo adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Isolamento e caratterizzazione di cellule endoteliali sinusoidali primarie del fegato di topo

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

Isolamento delle cellule primarie dell'epitelio pigmentato retinico del topo

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...