We thank Mr. O. Rios for his technical assistance.

Questo studio è stato approvato per l&#39;uso di materiale umano da parte dei comitati etici locali dell&#39;istituto di cura e il Centro ricerche e studi di avanzamento del Politecnico Nazionale. 1. Acquisizione tumore del campione <ol><li> Ottenere la biopsia del tessuto adenoma pituitario da un paziente attraverso una procedura chirurgica trans-sfenoidale 14. </li><li> Raccogliere il campione adenoma pituitario e metterlo in una bottiglia di vetro sterile contenente 50 ml di fresco 199 terreno di coltura (M199) arricchito con siero 10% fetale bovino (FBS), 0,6 mg / bicarbonato di sodio ml, 2,4 mg / ml di HEPES e l&#39;1% antibiotici (penicillina / streptomicina) a pH 7.4. Mantenere il campione in una bottiglia di vetro su ghiaccio. Nota: Il tessuto tumorale può essere elaborato diverse ore dopo l&#39;intervento chirurgico. </li><li> Trasportare il campione del tumore al laboratorio in bottiglia di vetro su ghiaccio. </li></ol> 2. Preparazione dei enzimatici Tissue dissociazione Solutions,Soluzione inibitore della tripsina e le soluzioni di alginato Nota: Prima della procedura di coltura cellulare Preparare le seguenti soluzioni. Filtrare tutte le soluzioni usando un filtro di 0,22 micron a membrana, prima dell&#39;uso. <ol><li> Preparare collagenasi soluzione all&#39;1% sciogliendo 5 mg di collagenasi (tipo I) in 5 ml di connessione M199-siero. </li><li> Preparare tripsina soluzione 0,01% sciogliendo 0,01 g di tripsina in 10 ml di PBS-EDTA 0,3%. </li><li> Preparare 0,1% soluzione di inibitore della tripsina sciogliendo 2 mg di inibitore di soia tripsina in 20 ml di libera M199-siero. </li><li> Preparare la soluzione DNAsi sciogliendo 20 ml di DNAsi I 134 U / ml in 4 ml di soluzione di inibitore della tripsina. </li><li> Preparare la soluzione EGTA (1.2 M) sciogliendo 22,8 mg di EGTA in 40 ml di 155 mM NaCl. Regolare il pH a 7,4. Continuare ad un volume finale di 50 ml. </li><li> Preparare la soluzione HEPES (20 mM) sciogliendo 238 mg HEPES in 50 ml di 155 mM NaCl. </li><li> Preparare la soluzione di alginato 1,2% sciogliendo 1.2g di sodio alginato in 100 ml di soluzione di HEPES. Autoclave la soluzione di alginato. </li><li> Preparare la soluzione di calcio (102 mm), sciogliendo 750 mg di CaCl 2 in 50 ml di soluzione HEPES, aggiustare il pH a 7,4 </li></ol> 3. cellulare primaria, Cultura e alginato incapsulamento <ol><li> All&#39;interno di una cappa a flusso laminare, posizionare il tessuto tumorale in una capsula di Petri 35 millimetri contenente circa 20 ml di PBS Ca 2+ e Mg 2+ libero, pH 7,4 (PBS *). </li><li> Prendere il tessuto con una pinzetta chirurgica e metterlo in un altro piatto di Petri contenente PBS fresco *, lavare delicatamente il tessuto. Ripetere questa operazione fino a quando i globuli rossi e detriti sono stati rimossi. </li><li> Utilizzando una pinzetta chirurgica, tenere il tessuto adenoma pituitario, e con una piccola forbice chirurgica, tagliato in piccoli pezzi. Con una pipetta Pasteur con bordi arrotondati, trasferire i frammenti di tessuto e il PBS * in una provetta da 15 ml, Centrifugare a 68 xg per 10 min a RT. Nota: per arrotondare i bordi di una Pasteurpipetta, scarsamente fiamma la punta della pipetta in un becco Bunsen. </li><li> Rimuovere il PBS * usando una pipetta Pasteur e aggiungere 3-5 ml della soluzione di collagenasi (circa 5 ml il 65 mm 3 di tessuto), mescolare due o tre volte per inversione. Incubare il tessuto in collagenasi per 30 min a 37 ° C in rotazione costante. </li><li> Centrifugare la provetta a 68 xg per 10 min a RT e rimuovere la collagenasi con una pipetta Pasteur. </li><li> Aggiungere 5 ml di DNAsi e mescolare due o tre volte invertendo, quindi centrifugare la provetta a 68 xg per 10 min a RT. Nota: Se il tessuto non è completamente dissociato, eseguire un secondo digestione enzimatica con tripsina per 3 minuti a 37 ° C. Arrestare la digestione con l&#39;aggiunta di 2 volumi di soluzione inibitore della tripsina. </li><li> Aspirare il surnatante con una pipetta Pasteur e scartare. Risospendere il pellet in 1 volume di soluzione EGTA e mescolare con 2 volumi di soluzione di alginato. </li><li> Mescolare delicatamente la sospensione cellulare e alginatopipettando con una pipetta Pasteur. Prendere una siringa sterile con un ago G 21, togliere il pistone; utilizzare una pipetta per caricare la siringa con la soluzione di alginato e cellule. </li><li> Inserire delicatamente lo stantuffo nella siringa e accuratamente erogare goccia a goccia la soluzione di alginato di cellule in un bicchiere di vetro contenente circa 30 ml di soluzione ricca di calcio. </li><li> Inserire l&#39;ago della siringa circa 5 cm fino alla soluzione di CaCl 2. I gel sospensione alginato cellule a contatto con il CaCl 2 per formare perle sferiche. Conservare le perline alginato di calcio nella soluzione per 5 min. Nota: Mescolare il bicchiere di vetro in cui sono sceso le perle. Spostare lentamente con la mano in movimenti circolari, per evitare che aderiscano l&#39;uno all&#39;altro. </li><li> Rimuovere con attenzione la soluzione di calcio con una pipetta Pasteur, e lavare le perle due volte con 3-5 ml di M199 arricchito con il 20% di FBS. </li><li> Trasferire le perline alginato in una cultura normale tessuto T25pallone di utilizzando una spatola sterile, aggiungere 4-5 ml di M199 arricchito con 20% FBS e incubati a 37 ° C in un incubatore umidificato con 5% di CO 2. Modificare il terreno di coltura ogni tre giorni. </li></ol> 4. Liberate cellule dal alginato Nota: Prima di liberare le cellule dalle perline alginato, cappotto 13 coprioggetto mm di diametro con poli-D-lisina. ATTENZIONE: ACIDO SOLFORICO è un acido altamente corrosivo, and Apts PUÒ CAUSARE OCCHI E DELLA CUTE. Utilizzare guanti in lattice per maneggiare i reagenti. <ol><li> In una cappa aspirante, con un punto pinzetta immergere i coprioggetti in un piatto di vetro Petri contenente il 20% acquosa di acido solforico per 1 ora a RT. </li><li> Decantare l&#39;acido solforico, e lavare i coprioggetti cinque volte con acqua distillata per 5 minuti ciascuna. Utilizzando un luogo punto pinzetta i coprioggetti puliti in una capsula di Petri contenente 0,1 M NaOH per 5 minuti a temperatura ambiente 15. </li><li> Decantare il NaOH e lavare una volta con distillareacqua ha portato, aspirare l&#39;acqua e asciugare i coprioggetti. </li><li> Utilizzare una micropipetta per rivestire i coprioggetti con APTS (amminopropil-trietossisilano) per 4 minuti (usare 500 microlitri per 6 lamelle) inondare la piastra di Petri con acqua distillata. aspirare rapidamente l&#39;acqua e continuare a lavare con acqua distillata, spostando i coprioggetti con una pinzetta point per permettere all&#39;acqua di penetrare al di sotto. </li><li> Aspirare l&#39;acqua e asciugare i coprioggetti, utilizzando un luogo punto pinzetta i coprioggetti in un piatto pulito Petri e sterilizzarli all&#39;interno del forno a microonde per 3 volte, 45 secondi ciascuno. Aggiungere 1,000 ml di acqua sterile e 100 mg di poli-D-lisina (0,1 mg / ml). </li><li> Incubare le coprioggetto con circa 50 ml di poli-D-lisina per 5 minuti a RT. Aspirare completamente i poli-D-lisina. Nota: Questo è importante perché solubile in poli-D-lisina nel mezzo di coltura può inibire la proliferazione cellulare. </li><li> Lavare i coprioggetti cinque volte con acqua sterile per 5 minuti ciascuno. Aspirare la stacqua e lasciare erile coprioggetto ad asciugare all&#39;interno di una cappa a flusso laminare. </li><li> Per analizzare il citoscheletro di actina, liberare le cellule ipofisarie dal alginato. Prendete un po &#39;delle perline alginato dal pallone di coltura usando un puntale 1 ml e metterli in un tubo da 15 ml. </li><li> Aggiungere 5 ml di citrato di sodio 55 mM (sciogliere 323,6 mg di Na 3 C 6 H 5 O 7, 2 H 2 O in 20 ml di soluzione HEPES) e centrifugare a 68 xg per 10 min a RT. Aspirare il surnatante e scartare. </li><li> Risospendere il pellet in 1 ml di caldo M199 arricchito con il 20% FSB; mescolare delicatamente la sospensione cellulare con una pipetta Pasteur. ATTENZIONE: falloidina E DAPI sono tossici. Usare i guanti per maneggiare i reagenti. </li><li> Per contare le cellule, trasferire 125 ml di 0,4% soluzione blu Trypan in una provetta da 1,5 ml. Aggiungere 75 ml di M199 e 50 ml di sospensione cellulare ottenuta nell&#39;ultimo passaggio (fattore di diluizione = 5). Mescolare accuratamente. </li><li> Utilizzare una punta micropipetta per place circa 10 microlitri della sospensione blue-cell Trypan nella camera emocitometro. </li><li> Contare le cellule nel contatore emocitometro utilizzando un microscopio invertito con illuminazione a contrasto di fase. Contare tutte le cellule nel centro della piazza 1 mm e quattro piazze d&#39;angolo 1 mm. Ottenere la densità delle cellule per millilitro moltiplicando il numero medio per quadrato per il fattore di diluizione -. 10 4 Per esempio, se i conteggi medi per quadrato è di 45 cellule x 5 x 104 = 2,25 x 106 cellule / ml. Nota: Contare le cellule in dieci piazze della camera di emocitometro. Non contare le cellule che toccano la linea mediana in basso e destra. </li><li> Seed 35.000 cellule su vetrini diametro di 13 mm rivestiti con poli-D-lisina. </li></ol> 5. Disposizione citoscheletro di actina <ol><li> Dopo 48 ore di cultura, rimuovere il terreno di coltura con una pipetta Pasteur e rapidamente aggiungere 500 microlitri per coprioggetto di PBS, immediatamente rimuovere la PBS. </li><li> Fissare e permeabilize tlui cellule con 500 microlitri per coprioggetto di tampone di fissaggio (0.1 M TUBI, pH 6,75, 4% PEG-6000, 1 mM EGTA, 1 mM MgSO 4, 0,5% Triton X-100 e 2% formaldeide) per 10 min a 37 ° C </li><li> Rimuovere il buffer fissazione e aggiungere 500 microlitri per coprioggetto del 3,5% paraformaldeide in PBS per 30 minuti a RT. Rimuovere la soluzione di paraformaldeide. </li><li> Lavare i coprioggetti per tre volte con l&#39;aggiunta di 500 microlitri per coprioggetto di PBS per 5 minuti ciascuna. Sciacquare i vetrini a temperatura ambiente, in costante agitazione. </li><li> Incubare le cellule con cloruro di ammonio 50 mM per 10 minuti, aspirare la soluzione di cloruro di ammonio e ripetere il punto 5.4. </li><li> Incubare le cellule con 1% di BSA (IgG-libera, proteasi-free) in PBS per 30 minuti, rimuovere la soluzione di BSA e ripetere il punto 5.4. </li><li> Incubare le cellule con 50 um di falloidina rodamina coniugato per 7 minuti a temperatura ambiente, ripetere il punto 5.4. </li><li> Incubare le cellule con 255 micron di DAPI diluito in PBS per 5 minuti a temperatura ambiente, ripetere il punto 5.4. Infine montare tegli coprioggetto con un mezzo di montaggio e di sigillare con smalto. </li><li> Acquisire le immagini delle disposizioni actina citoscheletro usando un microscopio confocale a scansione laser con un obiettivo 63x, a 541 nm di eccitazione lunghezza d&#39;onda. </li></ol>

<ol>
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The authors have nothing to disclose.

Questo protocollo ha diversi passaggi critici. Il primo è l&#39;omogeneità formato del branello, che è necessario per mantenere le stesse condizioni di diffusione dei nutrienti e gas in tutta la coltura cellulare. Nella nostra esperienza, l&#39;uso di un ago 21 G per rendere le perline alginato consente l&#39;acquisizione di una cultura efficace di formato del branello uniforme. Il secondo fattore importante è la distanza tra l&#39;ago; questa distanza deve essere non più di 5 cm dalla soluzione di calcio per evita...

Scaffold tridimensionali sono stati ampiamente utilizzati in coltura cellulare perché forniscono un sostegno strutturale tridimensionale per colture cellulari e la manutenzione di comunicazione 1 cellula-cellula. materiali polimerici Naturalmente derivati ​​sono stati utilizzati per fare impalcature tridimensionali contenenti collagene di tipo I, chitosano e alginato. Alginato è un polimero naturale derivato dalle alghe Macrocystispyrifera mare marrone (Kelp). Questo polimero è composto di unità ripetute di acido β-D-mannuronico (M) e α-L-guluronico acido (G) 2 e forma gel stabili in presenza di alcuni cationi bivalenti, come calcio e bario. (CaCl 2) soluzioni di cloruro di calcio vengono comunemente usati come reagente di reticolazione per formare perline alginato. Le soluzioni di alginato è sceso in CaCl 2 formare immediatamente gel sferici tridimensionali. Quando la soluzione di alginato viene miscelato con cellule, le cellule sono incapsulati in alginato bEADS 3. Alginato ha proprietà che hanno permesso di essere utilizzato come matrice per l&#39;incapsulamento di una varietà di cellule, comprese condrociti 4, mioblasti scheletrici 5 e cellule staminali neuronali 6. È un materiale inerte e permette la diffusione di nutrienti, ossigeno e prodotti metabolici che mantengono la sopravvivenza cellulare e la funzione; Inoltre, a differenza di altri sistemi di coltura a base di gel, cellule coltivate all&#39;interno perline alginato possono essere liberati dal scaffold usando chelanti del calcio, come citrato di sodio, e le cellule possono essere raccolte per ulteriori accertamenti 7. adenomi ipofisari sono in genere tumori benigni con tassi di proliferazione bassi. Linee cellulari adenoma pituitario di ratto sono state correttamente coltivate in un sistema a due dimensioni 8. Tuttavia, questa cultura di tumori a cellule dell&#39;ipofisi umane secrezione non è uno sviluppo efficiente; Le cellule tumorali ipofisarie disperse crescono poconella cultura, le cellule mostrano attaccamento limitata e la diffusione, e le cellule tipicamente formano aggregati galleggianti nel piatto cultura 9,10. Diversi tentativi sono stati fatti per ottenere una sospensione di cellule tumorali dell&#39;ipofisi, compreso un approccio enzimatica e dispersione meccanica 11, un approccio dispersione esclusivamente meccanica 12 e la coltura di espianti tumorali 10. Con questi approcci, diversi autori hanno ottenuto culture aderenti vitali per diversi periodi di tempo. Le capacità delle cellule tumorali secretorie di sopravvivere in questi sistemi bidimensionali dipendono dal tipo di tumore e loro tassi di proliferazione 9. Tuttavia, nelle culture a lungo termine, le cellule con fenotipo di fibroblasti predominano 11,13. Questo documento descrive un metodo per ottenere una coltura primaria di cellule d&#39;adenoma pituitario incapsulate in perline di alginato che li libera lontano dalla scaffold alginato che consente analisi dettagliateaspetti della loro biologia cellulare, ad esempio, le loro disposizioni del citoscheletro.

<table border="0" cellpadding="0" cellspacing="0" width="1019">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:401px;">Reagent&#160;</td>
			<td style="width:171px;"></td>
			<td style="width:143px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">199 culture medium&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>31100-027</td>
			<td>For culture, warm in a 37 &#176;C water bath before use&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum&#160;</td>
			<td>PAA</td>
			<td>A15-751</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium bicarbonate (NaHCO3)</td>
			<td>Merck</td>
			<td>106329</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-(2-Hydroxyethyl) piperazine-N&#180;-2ethanesulfonic acid (HEPES)</td>
			<td>Sigma&#160;</td>
			<td>H-4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>PAA</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS (Dulbecco&#39;s Phosphate Buffered Saline)</td>
			<td>Invitrogen&#160;</td>
			<td>21600-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase type I</td>
			<td>Worthington</td>
			<td>4176</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>27250-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Research Organics</td>
			<td>3002E</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sorbean trypsin inhibitor&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>17075-029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNAse</td>
			<td>Worthington</td>
			<td>2139</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alginic acid, From Macrocystis Pyrifera (Kelp)</td>
			<td>Sigma&#160;</td>
			<td>A-2158</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Calcium chloride (CaCl2)</td>
			<td>J.T. Baker</td>
			<td>1332</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium citrate (Na3C6H5O7)</td>
			<td>J.T. Baker</td>
			<td>3646</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piperazine-N,N&#8242;-bis(2-ethanesulfonic acid) (PIPES)</td>
			<td>Research Organics</td>
			<td>9624P</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene Glycol 6000 (PEG 6000)</td>
			<td>Calbiochem</td>
			<td>528877</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylene glycol tetraacetic acid (EGTA)</td>
			<td>Research Organics</td>
			<td>9574E</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnesium Sulfate (MgSO4)</td>
			<td>J.T. Baker</td>
			<td>2500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton-X 100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formaldehyde</td>
			<td>J.T. Baker</td>
			<td>2106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ammonium chloride (NH4Cl)</td>
			<td>J.T. Baker</td>
			<td>660</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA Bovine Serum Albumin IgG-Free</td>
			<td>Jackson Immuno Research</td>
			<td>001-000-161</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin&#8211;Tetramethylrhodamine B isothiocyanate</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Toxic. Use gloves to handle this reagent&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>Invitrogen&#160;</td>
			<td>D1306</td>
			<td>Toxic. Use gloves to handle this reagent&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sulfuric acid (H2SO4)</td>
			<td>J.T. Baker</td>
			<td>9681</td>
			<td>Highly corrosive. Use gloves to handle this reagent&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide (NAOH)</td>
			<td>Merck</td>
			<td>1.06498</td>
			<td>Can cause eye and skin irritation.Use gloves to handle this reagent&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(3-Aminopropyl)triethoxysilane (APTS)</td>
			<td>Sigma</td>
			<td>A-3648</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-D-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan blue solution</td>
			<td>Sigma</td>
			<td>T8154&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name&#160;&#160;</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment&#160;</td>
			<td></td>
			<td></td>
			<td>Toxic: May cause cancer. Use gloves to handle this reagent&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotator&#160;</td>
			<td>Boekel Scientific</td>
			<td>Model 230300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>DuPont Corporation&#160;</td>
			<td>Model sorvall TC6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">shaker&#160;</td>
			<td>Lab-line Instruments</td>
			<td>Model 314-820&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional alginate-bead culture of human pituitary adenoma cells

Un metodo di coltura tridimensionale è descritta in cui le cellule adenoma ipofisario primarie sono coltivate in perline alginato. Alginato è un polimero derivato da alghe brune mare. Brevemente, il tessuto tumorale è tagliato in piccoli pezzi e sottoposto ad una digestione enzimatica con collagenasi e tripsina. Successivamente, una sospensione cellulare viene ottenuta. La sospensione delle cellule tumorali viene miscelato con alginato di sodio 1,2% e rilasciato in una soluzione 2 CaCl, e la sospensione alginato / cellulare è gelificata a contatto con il CaCl 2 per formare perle sferiche. Le cellule incorporati nelle perle alginato sono forniti con nutrienti forniti dai terreni di coltura arricchito con 20% FBS. cultura tridimensionale in perline alginato mantiene la vitalità delle cellule adenoma per lunghi periodi di tempo, fino a quattro mesi. Inoltre, le cellule possono essere liberati dal alginato lavando le perline con citrato di sodio e seminate su vetrini per ulteriori analisi immunocitochimica. L&#39;uso di un celModello L cultura consente il fissaggio e la visualizzazione del citoscheletro di actina con il minimo disorganizzazione. In sintesi, perline alginato forniscono un sistema di coltura affidabile per il mantenimento delle cellule adenoma pituitario.

Questo protocollo è stato applicato con successo nella cultura e nella manutenzione delle cellule adenoma pituitario in perle di alginato per diversi periodi di tempo figura 1 mostra cellule incorporati in perline alginato dopo tre mesi.; queste cellule mostrano forme arrotondate bi-rifrazione sotto un microscopio ottico invertito (Figura 1). La Figura 2 mostra la localizzazione di N-caderina in cellule d&#39;adenoma pituitario di ratto...

Watch this Scientific Journal Video about Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells at JoVE.com

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensionale alginato-tallone coltura di cellule umane adenoma ipofisario

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

three-dimensional-alginate-bead-culture-human-pituitary-adenoma

Research

JoVE Journal

Medicine

9.9K Views.  Center for Research and Advanced Studies (CINVESTAV-IPN). Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Video: Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

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 ...

Ricerca

Medicina

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

Imaging tridimensionale di fibre nervose nocicettive intraepidermiche in biopsie cutanee umane

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Three-dimensional Tissue Culture modello per studiare Primario Osso Osseo e le sue Malignità

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Tre Culture dimensionali: strumento per studiare normale acinose Architettura vs trasformazione maligna delle cellule mammarie

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificazione del cancro al seno Cellula Invasività Utilizzando una tridimensionale (3D) Modello

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tridimensionale Co-cultura Modello di tumore stromale-Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionale (3D) Tumore Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Three-Dimensional Cultura Assay per esplorare Cancer Cell invasività e formazione di tumori Satellite

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Stampa tridimensionale di un'anomalia aortica complessa

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

Vertebroplastica percutanea percutanea percutanea (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...