Gli autori ringraziano M. Fiche di Ospedale universitario di Losanna per fornire la fotografia mastoplastica, M. Wirth e A. Ayyanan dell&#39;Istituto svizzero per la ricerca sperimentale sul cancro, Centro Nazionale di Competenza nella Ricerca Oncologia Molecolare, Facoltà di Scienze della Vita, Ecole Polytechnique Fédérale de Losanna per l&#39;assistenza tecnica e R. Clarke dell&#39;Università di Manchester per i commenti critici. La ricerca che ha portato a questi risultati ha ricevuto il sostegno di SNF3100A0-112090, Oncosuisse 531.817, e l&#39;iniziativa sui medicinali innovativi impresa comune nell&#39;ambito dell&#39;accordo di concessione n. 115.188, le risorse di cui si compone del contributo finanziario dal Settimo Programma Quadro dell&#39;Unione Europea (FP7 / 2007-2013) e la Federazione europea delle industrie e associazioni farmaceutiche aziende in contributi in natura. L&#39;indirizzo web di medicinali innovativi è <a href="http://www.imi.europa.eu/" target="_blank">http://www.imi.europa.eu/</a> . &lt;/ P&gt;

Considerazioni generali: prima del tempo, istituire un protocollo etico, preparano questionari per i pazienti, e vedere per la formazione del personale clinico. Prima di usare il materiale dalla chirurgia mastoplastica riduttiva, procurarsi le autorizzazioni, e garantire il consenso del paziente. Tissue può essere contagioso e deve essere gestito di conseguenza. Questo protocollo è stato approvato dal comitato etico di ISREC - Istituto svizzero di ricerca sperimentale sul cancro. 1. Tissue Recupero, lavorazione e Bio-banking Per garantire la qualità del campione ottimale per la bio-banking, una serie di misure vengono effettuate in ospedale prima del trasporto al laboratorio. All&#39;ospedale: <ol><li> Ottenere tessuti mammari umani in condizioni sterili da campioni di chirurgia mastoplastica riduttiva e sangue, se necessario. Tissue ha bisogno di essere esaminato da patologo che campione per escludere la presenza di lesioni maligne. </li><li> Posizionare la t senorilasciare sotto il cofano su una tavola sterili e preparare forbici, bisturi chirurgici e pinze che sono già sterilizzati con sterilizzatore a vapore. Fai taglio profondo in diversi siti del tessuto mammario con un bisturi per aprirlo. Nel tessuto mammario umano cercare filamenti bianchi, comprensivi di condotti e lobuli, che sono incorporati nel tessuto adiposo giallo. NOTA: Il rapporto tra il parenchimale bianco al tessuto adiposo giallo differisce tra le donne; in genere le donne più giovani hanno più tessuto epiteliale. </li><li> Scegli pezzi di materiale bianco e tenerli con una pinza, poi separarli delicatamente dal tessuto adiposo con le forbici. Evitare di prendere i vasi sanguigni. </li><li> Sciacquare pezzi di tessuto in PBS (PBS) pH 7,2 per rimuovere il sangue. Trasferire il tessuto di bottiglie di plastica da 250 ml contenenti mezzo F12 (Medium F12 Modified Eagle di Dulbecco) senza rosso con 2% di penicillina / streptomicina fenolo (comprese 5.000 unità / ml di penicillina e 5.000 g / ml di streptomicina) e l&#39;1% di unantibiotica / amfotericina B (comprese le 10.000 unità / ml di penicillina, 10.000 mg / ml di streptomicina, e 25 mg / ml di amfotericina B). Negozio tessuti nel mezzo per il trasporto al laboratorio. </li><li> Preparare pezzi di 3 - 5 mm di diametro per l&#39;estrazione di RNA e proteine ​​e flash congelarli in cryovials con il freddo (ghiaccio secco) isopentano. Trasferirli contenitore di ghiaccio secco per il trasporto al laboratorio. </li><li> Mettete i pezzi di 3-5 mm nel stampo e coprire con uno strato di Optimal taglio temperatura (OCT) e quindi inserire la muffa sul fondo piatto della scatola piena di isopentano freddo. Dopo aver completato il trasferimento di congelamento dello stampo alla casella ghiaccio secco. </li><li> Fissare 5 pezzi di 3 - 5 mm di diametro in paraformaldeide (PFA) 4% e altri 5 pezzi in formaldeide (FA) 4% in PBS a RT analisi istologica seguito. Due diversi fissativi sono raccomandati in alcuni anticorpi funzionano meglio con uno o l&#39;altro dei due. </li></ol> In laboratorio: <ol start="8"><li> Farecumento tutte le informazioni sulla mastoplastica riduttiva, data di chirurgia e se i campioni sono derivati ​​da sinistra contro mammella destra. </li><li> Posizionare i campioni congelati a -80 ° C e trasferire i tessuti fissi in cassette istologici dopo 2 ore di fissazione. Poi metterli sia in PBS o etanolo al 70%, per O / storage N a 4 ° C. Il giorno successivo, il trasferimento le cassette istologici alle strutture istologici per paraffina. </li></ol> 2. Tissue Digestione e stimolazione ormonale <ol><li> Trasferire le bottiglie contenenti i campioni di tessuto a un armadio flusso laminare in una camera di coltura cellulare P2. Mettere i pezzi di tessuto sul tagliere sterile (29 x 19 cm). Tenere il materiale con una pinza e raschiare il grasso e vasi rimanendo lontano dal parenchima mammario (parte bianca) con il bisturi. Posizionare il materiale bianco in PBS in un piatto di cultura 10 cm. </li><li> Dopo aver rimosso il materiale grasso da tutti i pezzi e disporli secondo la regola di sicurezzas del laboratorio, posizionare le parti bianche contenenti i tessuti epiteliali di nuovo sul tagliere. Utilizzando bisturi opposte tagliare il tessuto e tagliarla a pezzi di diametro fino a 2 mm. </li><li> Il materiale macinato in provette di dimensioni adeguate: fino a 1, 2,5 e 10 ml in un 5, 15 o 50 ml tubi, rispettivamente. Tipicamente a 5 ml rendimenti tubo di materiale sufficiente per paraffina embedment e ormonali stimoli, e 15 ml produce tubo di materiale sufficiente per fluorescenza cellulare Attivato (FACS). Tubi più grandi sono utilizzati per il congelamento aliquote. </li><li> Preparare master mix digestione composto di media (DMEM / F12 senza rosso fenolo) con 1% di penicillina / streptomicina / amfotericina B e collagenasi A (Clostridiopeptidase A da Clostridium histolyticum). Dose collagenasi secondo i tempi di digestione. <ol><li> Per 12, 24, e 36 h di incubazione, utilizzare concentrazione finale di collagenasi di 1,5 mg / ml, 1,0 mg / ml e 0,5 mg / ml. Aggiungere mix digestione padrone fino a 4 volte ilvolume del materiale digerito per garantire un&#39;adeguata agitazione e aerazione. NOTA: A seconda del disegno sperimentale e la durata della stimolazione ormonale, aggiungere ormoni durante o dopo la digestione enzimatica fase e microstruttura recupero. </li></ol></li><li> Aggiungere ormone (s) alla concentrazione desiderata per testare campioni ed il veicolo al controllo. Per la stimolazione con 17-β-estradiolo, e promegestone (R5020) utilizzare una concentrazione finale di 20 nM. NOTA: R5020 è un agonista del recettore del progesterone sintetica, che è più stabile del progesterone naturale in mezzo. <ol><li> Porre le provette del mixer rullo interno un incubatore a 37 ° C e 40 rpm O / N. Prima di mettere i campioni del mixer, li risospendere molto bene per garantire che tutto il materiale distribuisce lungo le provette. </li></ol></li><li> Come digestione richiede almeno 12 ore (O / N), per tempi di stimolazione ormonale inferiori a 12 ore, aggiungere gli ormoni dopo il recupero delle microstrutture (decoda sotto il punto 3). Aggiungere gli ormoni, come si sospendere nuovamente le microstrutture e posizionare la sospensione su piastre di fissaggio ultra-basse. Microstrutture tessuti possono essere conservati su piastre di fissaggio ultra-bassi per un massimo di 7 giorni in mezzo di crescita-libera-factor di conservazione di circa il 70% della redditività. </li><li> Per RNA e l&#39;analisi delle proteine, raccogliere microstrutture tessuti per centrifugazione a 400 xg per 5 minuti e flash freeze in provette. </li></ol> 3. Recupero di microstrutture del tessuto <ol><li> Centrifugare il tubo (s) a 400 xg per 5 min. Aspirare il grasso dalla parte superiore della provetta e trasferire il supernatante del surnatante acquoso rimanente, che si arricchisce in fibroblasti, in un provetta da centrifuga e centrifugare a 1250 xg per 5 min. NOTA: le microstrutture del tessuto del seno sono molto appiccicoso, per tutte le fasi successive risospendere il pellet agitando delicatamente il tubo. Pipettaggio non è raccomandato a causa del rischio di perdere materiale. </li&gt; <li> Risospendere il pellet arricchiti in microstrutture ed in fibroblasti derivati ​​da surnatante acquoso in PBS, 2% Fetal Calf Serum (FCS). Successivamente girare entrambi i tubi a 1.250 xg per 5 minuti e poi scarta il surnatante. </li><li> Risospendere il pellet in 3 - 5 ml rosso tampone di lisi del sangue cellulare e trasferirli a pulire i tubi. Li Incubare per 5 minuti a RT. </li><li> Aggiungere 2x volume di PBS, 2% FCS e centrifugare a 1250 xg per 5 min. Lavare il pellet con PBS, 2% FCS e centrifugare a 1250 xg per 5 min. Ripetere questa operazione due volte. </li></ol> 4. I campioni di Citometria a Flusso <ol><li> Aggiungere 1 - 2 ml di tripsina 0,25% al ​​pellet e mescolare pipettando su e giù delicatamente per 2 minuti con una Pipetman 1.000 microlitri a dissociarsi cellule. </li><li> Inibire l&#39;attività tripsina aggiungendo 9 ml PBS, 2% FCS. Centrifugare a 450 xg per 5 min e risospendere il pellet in 10 ml di PBS, 2% FCS. </li><li> Trasferire la sospensione cellulare su un filtro cella 40 micron posto su top di un tubo da centrifuga da 50 ml per preparare singole cellule. Se pochi microlitri di sospensione cellulare rimangono sulla sommità del filtro, aiutare passano attraverso il filtro da essi aspirando dall&#39;altro lato del filtro con una pipetta e aggiungere alle microstrutture filtrati. </li><li> Cellule immunitarie separati, fibroblasti e cellule endoteliali di microstrutture con un cocktail di anti-CD45, anti-FAP e anti-CD31 anticorpi e poi ordinare con FACS, sulla base di pan-epiteliale EpCAM marcatore CD10 e di separare le cellule luminali e basali, rispettivamente 10 . </li></ol> 5. I campioni di Istologia <ol><li> Dopo aver lavato il materiale meccanicamente enzimaticamente digerito come sopra descritto, fissare pellets da 1,5 ml materiale iniziale in 1 ml di 4% PFA in PBS a temperatura ambiente per 30 min. </li><li> Centrifugare a 1.250 xg per 5 minuti e lavare con 1-2 ml di PBS due volte. </li><li> Centrifugare a 16.000 xg per 10 min. Durante questo tempo, preparare 1% polivalente agarosio in Tris-acetato-EDTA (TAE) tampone e riscaldare la soluzione nel forno a microonde fino a quando la soluzione viene a ebollizione. <ol><li> Rimuovere la soluzione dal forno a microonde e agitare il matraccio delicatamente per miscelare la soluzione e risospendere le particelle rimanenti agarosio. Soluzione di agarosio Raffreddare a 50 ° C e versare 1 ml in uno stampo di base di paraffina dimensionato 31 × 23 × 13,5 millimetri. </li></ol></li><li> Eliminare il supernatante e posizionare i pellet sopra dello strato solidificato (1 - 3 mm) di agarosio utilizzando una spatola con un&#39;estremità piatta. Tutti Coprire con un ulteriore strato di agarosio (40 ° C). </li><li> Dopo le solidifica agarosio, di solito entro 5 minuti, mettere i blocchi agarosio nelle cassette per istologia paraffina incastro. Insieme con i campioni di agarosio posto un ufficio carta bianca nella cassetta con tutte le informazioni sul numero mastoplastica riduttiva, condizione sperimentale, e la data scritta in lettere minuscole a matita; questa scrittura resiste tutte le fasi di trattamento successive. </li><li> Mettere le cassette inPBS O / N, o in 70% di etanolo per immagazzinaggio durante il fine settimana, a 4 ° C. Non abbiamo notato alcuna differenza tra le due soluzioni di memorizzazione. Poi il trasferimento cassette istologici alle strutture istologici per paraffina. </li></ol> 6. microstrutture congelamento (Future) la coltura <ol><li> Risospendere microstrutture del tessuto a congelamento mezzo costituito da FCS con il 10% dimetilsolfossido (DMSO) e congelare 1 ml aliquote in cryovials. Il numero di cryovials ottenuti dipende dalla quantità di materiale primario ottenuto da mastoplastica riduttiva. </li><li> Bloccare le cryovials contenenti microstrutture tessuto utilizzando un tasso freezer controllata con il seguente programma: 5 min dimora a 4 ° C, diminuendo la temperatura a -7 ° C con velocità di 6 ° C / min, diminuendo la temperatura a -12 ° C con velocità di 6 ° C / min, 10 min dimora, diminuendo la temperatura a -40 ° C con velocità di 6 ° C / min, diminuzione della temperatura a -80 ° C convelocità di 6 ° C / min. Poi trasferire i cryovials a -80 ° C freezer. </li><li> Il giorno dopo, trasferire i cryovials da -80 ° C a serbatoio di azoto liquido per la conservazione a lungo. </li></ol>

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Gli autori non hanno nulla da rivelare.

La cultura ex vivo qui descritto fornisce microstrutture del tessuto del seno contenenti dotti e lobuli intatte, insieme ad altri tipi di cellule che normalmente si trovano in seno femminile umano. Nella lavorazione del tessuto mammario umano normalmente ottenuti da mammoplastiche riduzione, rimozione del tessuto adiposo, la digestione enzimatica e meccanica della matrice stromale, e lisi dei globuli rossi arricchisce per frammenti condotto latte e unità lobulare duttali terminali. Digestione enzimatica Gentle...

A correction was made to <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>. There was an error with the author, Marie Shamsheddin&#39;s, name. The author&#39;s name has been corrected to:
Marie Shamseddin
instead of:
Marie Shamsheddin

A correction to the author list has been made for the article <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>.

Erratum: An Ex Vivo Model to Study Hormone Action in the Human Breast

Informazioni sul paesaggio mutazionale di cancro al seno sta aumentando a ritmi sostenuti. Meno attenzione è stata rivolta a fattori sistemici che influenzano lo sviluppo del cancro al seno. L&#39;esposizione agli ormoni riproduttivi ha un impatto importante sulla progressione della malattia 1-3. Tuttavia, i meccanismi con cui gli ormoni riproduttivi incidono sul seno umano sono poco conosciuti. Lavora con modelli di topo geneticamente ha rivelato che coinvolgono cellule segnalazione intrinseca e paracrine attraverso diversi effettori a valle 4. La conoscenza limitata su azione ormonale nel seno umano è in gran parte attribuibile alla mancanza di modelli adeguati. Maggior parte dei lavori sui meccanismi di recettore per gli estrogeni (ER) e del recettore del progesterone (PR) segnalazione è stata eseguita con linee di cellule del recettore dell&#39;ormone positivi cancro al seno, come MCF-7 e T47D. Questi sono stati ottenuti da versamento pleurico di pazienti con carcinoma mammario avanzato che aveva already ricevuto trattamenti multipli 5. La rilevanza biologica dei risultati di tali modelli semplici in vitro del seno umano è geni discutibili e bersaglio identificati in questi modelli in vitro sono differiscono da geni bersaglio che sono identificati in modelli animali 6. Quando le cellule epiteliali mammarie umane primaria sono coltivati ​​in vitro tendono a perdere l&#39;espressione del recettore ormonale e, quindi, la risposta ormonale 7,8. Questo problema può essere circumventd da approcci 3D sofisticati con matrigel. In questo modo, C. Clarke e colleghi sono riusciti a stabilire cellule epiteliali mammarie che hanno mantenuto l&#39;espressione del recettore ormonale e hanno mostrato una risposta proliferativa di progesterone stimolazione 9. Tuttavia, due importanti in vivo dei recettori del progesterone geni bersaglio, Wnt-4 e RANKL, non sono state indotte dopo stimolazione progesterone in questo sistema 9. Questo approccio è stato recentemente approvato ulteriormente con in vitro </em&gt; ormone pretrattamento e induzione RANKL è stato raggiunto 10. Un avvertimento resta che matrigel ha attività che dipendono in batch, è costoso, e richiede un disegno sperimentale che è adatto solo per il numero di cellule piccole. Sulla base della constatazione che in ER vivo e segnalazione PR sono in gran parte mediata da interazioni paracrine 11, abbiamo sostenuto che le interazioni intercellulari devono essere mantenuti. Un altro fattore importante che si perde come tessuti sono correlate alle cellule singole per la cultura in vitro sono le interazioni delle cellule epiteliali con la matrice extracellulare; ma questi sono cruciali per la differenziazione epiteliale e la loro distruzione è importante nella tumorigenesi 12. Con questo in mente, abbiamo stabilito un metodo per isolare microstrutture del tessuto mammario da fresco materiale chirurgico scarto 13. Il parenchima mammario, costituito da un epitelio doppio strato con luminale interna ed esterna myoepithelicellule al, viene sezionato dal tessuto adiposo e sottoposti alla dissociazione enzimatica e meccanica. Dopo il lavaggio e centrifugazione, frammenti di condotti del latte si ottengono che mantengono una stretta interazione con molte cellule stromali. Queste microstrutture del tessuto rimangono ormone reattivo. Il modello è stato validato in campioni clinici 13. Come tale, il presente procedimento può aiutare a studiare l&#39;azione ormonale nel seno in un contesto biologicamente e clinicamente rilevante.

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		<tr height="25">
			<td height="25" style="height:25px;width:335px;">CO2 incubator</td>
			<td style="width:164px;">Hera Cell Heraeus</td>
			<td style="width:119px;">51022391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Roller Mixer SRT9D</td>
			<td style="width:164px;">Sturt</td>
			<td style="width:119px;">SRT9D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Histology Cassettes</td>
			<td style="width:164px;">Medizintechnik</td>
			<td style="width:119px;">81-0021-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cell Strainer</td>
			<td style="width:164px;">Falcon</td>
			<td style="width:119px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Strile Surgical Blade&#160;</td>
			<td style="width:164px;">Aesculap</td>
			<td>BB536&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Scalpel</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BB084R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Forceps</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BD047R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Scissors</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BC374R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Paraffin base mold</td>
			<td style="width:164px;">SAKURA</td>
			<td style="width:119px;">4166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Optimal Cutting Temperature (OCT) Cryomatrix</td>
			<td style="width:164px;">Thermoscientific</td>
			<td style="width:119px;">6769006</td>
			<td style="width:260px;">Fetal Bovine Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Antibiotic/Antimycotic</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DMEM F/12</td>
			<td style="width:164px;">Life Technologies</td>
			<td>11039-021</td>
			<td>Prewarm (37&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Collagenase</td>
			<td style="width:164px;">Roche</td>
			<td style="width:119px;">11 088 793 001</td>
			<td>Prewarm (37&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fetal Bovine Serum</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">10270</td>
			<td>Can be replaced with Fetal Calf Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cell Blood Lysis Buffer</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">R7757</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Trypsin-EDTA</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Agarose</td>
			<td style="width:164px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Formaldehyde</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">F1635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Paraformaldehyde</td>
			<td style="width:164px;">Roth</td>
			<td style="width:119px;">335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Isopentane</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">M32631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethanol</td>
			<td style="width:164px;">Merck</td>
			<td style="width:119px;">1009831000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cryogenic vials (5.0ml)&#160;</td>
			<td style="width:164px;">VWR, International</td>
			<td style="width:119px;">479-0820</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Ultra low attachment culture dish</td>
			<td style="width:164px;">Corning, NY 14831</td>
			<td style="width:119px;">3471</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo model to study hormone action in the human breast

Lo studio di azione dell&#39;ormone nel seno umano è stata ostacolata dalla mancanza di adeguati sistemi modello. Alla cultura in vitro, cellule epiteliali mammarie primarie tendono a perdere l&#39;espressione del recettore dell&#39;ormone. Ampiamente recettore ormone linee di cellule di cancro al seno positivo utilizzati sono di scarsa rilevanza per la situazione in vivo. Qui, descriviamo un modello ex vivo per studiare l&#39;azione di ormoni nel seno umano. Campioni di tessuto mammario umano fresco da materiale di scarto chirurgico quali mammoplastiche riduzione o mammectomies sono meccanicamente ed enzimaticamente digerite per ottenere frammenti di tessuto contenenti dotti e lobuli e molteplici tipi di cellule stromali. Queste microstrutture tessuti conservati in terreno di base senza fattori di crescita mantenere i contatti intercellulari, l&#39;architettura dei tessuti, e rimangono ormone reattivo per diversi giorni. Essi sono facilmente elaborati per l&#39;RNA e l&#39;estrazione di proteine, l&#39;analisi istologica o memorizzati in congelamento medio. Fluorescenzaattivato ordinamento delle cellule (FACS) può essere utilizzato per arricchire per specifiche popolazioni cellulari. Questo protocollo offre un approccio standard semplice per gli studi traslazionali con altamente complessi, variegati campioni umani.

Per studiare il ruolo di estrogeni e progesterone e per capire meglio le loro funzioni molecolari nel seno umano, raccogliamo campioni freschi umani del tessuto del seno da pazienti sottoposti mammoplastiche riduzione (Figura 1) dopo aver ottenuto il loro consenso informato. Abbiamo anche ottenere medica e riproduttiva storia del paziente, così come un campione di sangue per determinare i livelli sierici di progesterone al momento dell&#39;intervento. Tissue da mammoplastiche riduzione freschi è mecca...

Watch this Scientific Journal Video about An Ex vivo Model to Study Hormone Action in the Human Breast at JoVE.com

An Ex vivo Model to Study Hormone Action in the Human Breast

Abbiamo sviluppato un nuovo modello di ex vivo per studiare l&#39;azione di ormoni nel seno umano. Si basa su microstrutture tessuti isolati da campioni di tessuto del seno chirurgici che conservano l&#39;architettura del tessuto, le interazioni intercellulari, e segnalazione paracrina.

Un<em&gt; Ex vivo</em&gt; Modello per studiare Hormone azione in seno umano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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Research

JoVE Journal

Medicine

An Ex vivo Model to Study Hormone Action in the Human Breast

11.3K Views.  Ecole polytechnique fédérale de Lausanne.  Abbiamo sviluppato un nuovo modello di ex vivo per studiare l'azione di ormoni nel seno umano. Si basa su microstrutture tessuti isolati da campioni di tessuto del seno chirurgici che conservano l'architettura del tessuto, le interazioni intercellulari, e segnalazione paracrina. 

Video: An Ex vivo Model to Study Hormone Action in the Human Breast

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Ricerca

Medicina

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Un umano<em&gt; Ex Vivo</em&gt; Placca aterosclerotica modello per studiare Lesione Biologia

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Umana Dupuytren di<em&gt; Ex Vivo</em&gt; Cultura per lo Studio dei miofibroblasti e matrice extracellulare Interazioni

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Metodi per la coltura di femore umano espianti di tessuto di studio sul carcinoma della mammella cellulare colonizzazione della nicchia metastatico

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In vivo ed ex vivo approcci per studiare il cancro ovarico metastatico colonizzazione delle strutture Lattea Spot in peritoneale adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Latte materno aumenta la crescita di Enteroids: un modello Ex Vivo di proliferazione delle cellule

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...