Questa ricerca è stata supportata dalla "Istituzione di standard di misurazione per la chimica e le radiazioni", numero di sovvenzione KRISS-2020-GP2020-0003, e "Sviluppo di standard di misurazione e tecnologia per biomateriali e convergenza medica", numero di sovvenzione KRISS-2020-GP2020-0004 programmi, finanziati dal Korea Research Institute of Standards and Science. Questa ricerca è stata sostenuta anche dal Ministero della Scienza e delle TIC (MSIT), dalla National Research Foundation of Korea (NRF-2019M3A9F3065868), dal Ministero della Salute e del Welfare (MOHW), dal Korea Health Industry Development Institute (KHIDI, HI20C0558), dal Ministero del Commercio, dell'Industria e dell'Energia (MOTIE) e dal Korea Evaluation Institute of Industrial Technology (KEIT, 20009350). ID ORCID (Hee Min Yoo: 0000-0002-5951-2137; Dukjin Kang: 0000-0002-5924-9674; Seil Kim: 0000-0003-3465-7118; Joo-Eun Lee: 0000-0002-2495-1439; Jina Lee: 0000-0002-3661-3701). Ringraziamo Chang Woo Park per l'assistenza con gli esperimenti.

1. Colture cellulari batteriche e preparazione di Lactobacillus cell-free surnatante (LCFS)
NOTA: I passaggi 1.2 – 1.9 sono condotti in una camera anaerobica.
<ol><li>Preparare una piastra di agar MRS e un brodo contenente L-cisteina e sterilizzare in autoclave.</li>
	<li>Pre-incubare la piastra di agar MRS in camera anaerobica H2 mantenuta a 37 °C con 20 ppm di ossigeno.</li>
	<li>Scongelare lo stock batterico di Lactobacillus e inoculare la piastra di agar con la coltura batterica (Figura 1A (i)).</li>
	<li>Incubare i batteri per 2 - 3 giorni in camera anaerobica H2 a 37 °C e 20 ppm di ossigeno fino ad ottenere singole colonie batteriche.</li>
	<li>Lavare e asciugare il tubo di coltura anerobica di tipo Hungate. Autoclave del tubo di coltura a 121 °C per 15 min.</li>
	<li>Quindi incubare il tubo in camera anaerobica H2 a 37 °C e 20 ppm di ossigeno per rimuovere l'ossigeno.</li>
	<li>Posizionare 2 - 3 ml di brodo MRS nel tubo. Sigillare il tubo con un tappo di gomma butilica e avvitare il cappuccio.</li>
	<li>Ottenere una singola colonia con un anello e posizionarla nel tubo di coltura da 1,5 ml con 500 μL di 1x PBS. (Figura 1A (ii)).</li>
	<li>Sospendere la colonia utilizzando una siringa da 1 mL (Figura 1A (iii)). Farlo inserendo l'ago della siringa da 1 mL al centro del coperchio del tubo, aspirando la colonia sospesa e quindi riutilizzandola nel mezzo di brodo MRS. (Figura 1A (iv)).</li>
	<li>Incubare il mezzo di brodo MRS in un incubatore shaker per 2 giorni (37 °C, 5% CO2, 200 rpm).</li>
	<li>Misurare la densità ottica (OD) utilizzando uno spettrofotometro per monitorare le curve di crescita batterica fino a quando l'assorbanza a OD620 raggiunge 2.0.</li>
	<li>Separare il pellet batterico e il mezzo condizionato centrifugando a 1.000 x g per 15 min. Lavare i pellet batterici raccolti con 1x PBS e riconsesporre in 4 ml di RPMI 1640 integrato con il 10% di siero bovino fetale. Non includere antibiotici nel mezzo.</li>
	<li>Mantenere i pellet batterici in RPMI e incubare in un incubatore shaker per 4 ore a 37 °C con il 5% di CO2 ad una velocità di 100 rpm.</li>
	<li>Per la preparazione del surnatante probiotico, rimuovere il pellet batterico tramite centrifugazione a 1000 x g,per 15 min a 4 °C. Filtrare sterile il surnatante recuperato utilizzando un filtro da 0,22 μm e conservare a -80 °C fino all'uso.</li>
</ol>2. Generazione di sferoidi
<ol><li>
Preparazione delle linee cellulari del cancro del colon-retto
	<ol>
<li>Coltivare linee cellulari DLD-1, HT-29 e WiDr come monostrati fino al 70-80% di confluenza e incubare la piastra a 37 °C in un incubatore co2 al 5% (terreno di crescita: RPMI contenente il 10% di siero bovino fetale (FBS) e l'1% di penicillina-streptomicina).</li>
		<li>Per le cellule coltivate in una capsula di Petri da 100 mm, lavare la piastra due volte con 4 ml di 1x PBS. Aggiungere 1 mL di tripsina-EDTA allo 0,25% e incubare la capsula di Petri per 2 minuti a 37 °C in un incubatore a CO2 al 5% per dissociare le cellule.</li>
		<li>Dopo l'incubazione, verificare la dissociazione cellulare al microscopio e neutralizzare la tripsina-EDTA con 5 ml di terreno di coltura.</li>
		<li>Trasferire le cellule dissociate in un tubo conico da 15 ml e centrifugare per 3 minuti a 300 x g.</li>
		<li>Scartare il surnatante e risospentare delicatamente con 3 ml di terreno di crescita.</li>
		<li>Contare le cellule con tripano blu per determinare le cellule vitali usando un emocitometro. (Figura 1B (i))</li>
	</ol>
</li>
	<li>
Formazione sferoide
	<ol>
<li>In un tubo conico da 15 mL, diluire le cellule da 2.1.5 per ottenere 1 - 2 x 105 celle/mL (Figura 1B (ii))</li>
		<li>Aggiungere la concentrazione finale dello 0,6% di metilcellulosa alla sospensione cellulare e trasferire le cellule diluite in un serbatoio sterile. NOTA: Per ogni linea cellulare, la quantità di metilcellulosa necessaria deve essere titolata e determinata di conseguenza.</li>
		<li>Utilizzare una pipetta multicanale per erogare 200 μL di celle a ciascun pozzera di una micropiastra a fondo tondo a 96 pozzette con attacco ultra-basso. (Figura 1B (iii))</li>
		<li>Incubare la piastra a 37 °C in un incubatore a CO2 al 5% per 24 - 36 ore.</li>
		<li>Dopo 24 - 36 ore, osservare la piastra al microscopio ottico per garantire la formazione di sferoidi.</li>
	</ol>
</li>
</ol>3. Trattamento delle cellule tumorali del colon-retto 3D con LCFS
<ol><li>Generare sferoidi come descritto nei passaggi 2 e 3.</li>
	<li>Prima di eseguire il trattamento LCFS, scongelare l'LCFS congelato a temperatura ambiente (RT) per 10 - 20 min.</li>
	<li>Inoculare la soluzione stock LCFS in un mezzo di crescita. Diluire in serie al 25%, 12,5% e 6% nel mezzo di crescita (cioè 25% LCFS = 150 μL di terreno di crescita + 50 μL di LCFS).</li>
	<li>Estrai la piastra di coltura cellulare contenente sferoidi dall'incubatore e rimuovi il più possibile il mezzo di crescita da ciascun pozzo usando una pipetta da 200 μL.</li>
	<li>Aggiungere il mezzo di crescita con LCFS sulle cellule e incubare a 37 °C in un incubatore CO2 al 5% per 24 - 48 ore. NOTA: il volume da utilizzare dipenderà dalla dimensione della piastra come segue: 2 ml per piastre di coltura cellulare a 6 pozzi; 200 μL per piastre di coltura cellulare a 96 pozzi.</li>
</ol>4. Vitalità cellulare per sferoidi
<ol><li>Preparare 8 - 10 sferoidi del cancro del colon-retto trattati con LCFS in piastre multi-pozzo a parete opaca (i saggi di vitalità cellulare vengono eseguiti 48 ore dopo il trattamento con LCFS).</li>
	<li>Scongelare il reagente di vitalità cellulare (vedi Tabella dei materiali)a 4 °C per la notte.</li>
	<li>Equilibrare il reagente di vitalità cellulare a temperatura ambiente prima dell'uso.</li>
	<li>Prima di eseguire il test, rimuovere il 50% dei mezzi di crescita dagli sferoidi.</li>
	<li>Aggiungere 100 μL di reagente di vitalità cellulare a ciascun pozzo. NOTA: il volume da utilizzare dipenderà dalla dimensione della piastra come segue: 100 μL per piastre di coltura cellulare a 96 pozzi.</li>
	<li>Mescolare vigorosamente il reagente per 5 minuti per promuovere la lisi cellulare.</li>
	<li>Incubare per 30 min – 2 h a 37 °C.</li>
	<li>Registrare la luminescenza.</li>
</ol>5. Analisi quantitativa in tempo reale della reazione a catena della polimerasi per gli sferoidi
<ol><li>Per ogni condizione, preparare 10 - 15 sferoidi in un tubo da 2 ml e centrifugare per 3 minuti a 400 x g.</li>
	<li>Scartare il surnatante e lavare gli sferoidi due volte in 1 mL di PBS 1x ghiacciato. NOTA: Evitare la centrifugazione, lasciare che gli sferoidi si depositino.</li>
	<li>Aspirare il più possibile il PBS 1x e isolare l'RNA utilizzando un kit disponibile in commercio.</li>
	<li>Sintetizzare cDNA da 1 μg di RNA utilizzando un kit disponibile in commercio secondo il protocollo del produttore.</li>
	<li>Preparare un mix principale per eseguire tutti i campioni in triplice copia (vedere Tabella 1 e Tabella 2).</li>
	<li>Eseguire l'amplificazione in un mix master di modelli da 20 μL in ogni piastra qPCR.</li>
	<li>Mescolare bene le reazioni e girare se necessario.</li>
	<li>Eseguire campioni secondo le raccomandazioni del produttore dello strumento (Tabella 3).</li>
</ol>6. Western blotting da sferoidi
NOTA: Quando si raccolgono gli sferoidi, utilizzare una pipetta da 200 μL e tagliare l'estremità delle punte per evitare di disturbare la loro struttura.
<ol><li>Per ogni condizione, preparare 30 - 40 sferoidi in un tubo da 2 ml.</li>
	<li>Posizionare il tubo sul ghiaccio e lasciare che gli sferoidi si depositino sul fondo del tubo da 2 ml.</li>
	<li>Scartare il surnatante e lavare gli sferoidi due volte in 1 mL di PBS ghiacciato 1x NOTA: Evitare la centrifugazione, lasciare che gli sferoidi si depositino.</li>
	<li>Aspirare il più possibile il PBS 1x e aggiungere il tampone RIPA con un cocktail inibitore della proteasi (10 sferoidi = 30 μL di tampone RIPA).</li>
	<li>Lisire le cellule tubando su e giù ed eseguire la sonicazione per 30 s con 30 s di riposo sul ghiaccio per 10 cicli.</li>
	<li>Centrifugare i laccio proteica a 15000 x g per 15 min a 4 °C.</li>
	<li>Determinare la concentrazione proteica per ogni lizzata cellulare.</li>
	<li>Prima del caricamento, far bollire ogni llisi cellulare in un tampone campione a 100 °C per 10 minuti.</li>
	<li>Caricare quantità uguali di proteine nei pozzi del gel SDS-PAGE ed eseguire il gel per 1 - 2 ore a 100 V.</li>
	<li>Trasferire la proteina dal gel alla membrana PVDF.</li>
	<li>Dopo il trasferimento, bloccare la membrana per 1 ora a temperatura ambiente utilizzando un tampone di blocco (5% di latte scremato + TBS con 0,05% di Tween-20).</li>
	<li>Incubare la membrana con 1:1.000 diluizioni di anticorpi primari(Tabella 4)in 1x TBST con tampone BSA al 5% a 4 °C durante la notte.</li>
	<li>Lavare la membrana tre volte con TBST, 15 minuti per ogni lavaggio.</li>
	<li>Incubare la membrana con 1:2.500 diluizioni di anticorpi secondari nel tampone bloccante a temperatura ambiente per 2 ore (vedi Tabella dei materiali).</li>
	<li>Lavare la membrana tre volte con TBST, 15 minuti per ogni lavaggio.</li>
	<li>Preparare la membrana per il rilevamento HRP con un substrato chemiluminescente.</li>
	<li>Acquisisci immagini chemiluminescenti.</li>
</ol>7. Colorazione dello ioduro di propidio (PI) degli sferoidi
<ol><li>Preparare 5-10 sferoidi come descritto nella fase 4.1 e posizionare gli sferoidi in un incubatore a 37 °C e 5% CO2.</li>
	<li>Diluire uno stock di 1 mg/mL di PI 1:100 in 1x PBS.</li>
	<li>Rimuovere il 50% del mezzo da ciascun pozzo della piastra a 96 pozzi.</li>
	<li>Aggiungere 100 μL della soluzione PI a ciascun pozzo e posizionare i pozzi in un incubatore a 37 °C e 5% CO2 per 10 - 15 min.</li>
	<li>Lavare la soluzione PI con 1x PBS.</li>
	<li>Aggiungi 200 μL di terreno di coltura e scatta un'immagine usando un microscopio a fluorescenza. Analizza l'intensità della fluorescenza usando l'immagine J per ottenere il conteggio di vitalità dello sferoide.</li>
</ol>8. Analisi FACS degli sferoidi
<ol><li>Generare sferoidi come descritto in precedenza.</li>
	<li>Per ogni condizione, preparare 30 - 40 sferoidi in un tubo FACS e centrifuga per 3 minuti a 400 x g e RT.</li>
	<li>Aspirare il surnatante e lavare gli sferoidi in 3 mL di 1x PBS, quindi centrifugare a 400 x g per 3 min a 4 °C.</li>
	<li>Aspirare il surnatante e aggiungere 200 μL di 0,25% di tripsina-EDTA, quindi incubare a RT per 2 -3 min. NOTA: il tempo di incubazione dipende dalla dimensione dello sferoide e dal tipo di cella.</li>
	<li>Aggiungere 1 mL di tampone FACS e dissociare delicatamente gli sferoidi utilizzando una pipetta da 200 μL.</li>
	<li>Centrifugare le cellule dissociate a 400 x g e 4 °C per 3 min. NOTA: buffer FACS = 1x PBS + 2,5% FBS, filtrato utilizzando un filtro superiore da 0,22 μm.</li>
	<li>Eliminare il surnatante e aggiungere il reagente 7-AAD/Annexin V (7AAD (5 μL), Annexin V (5 μL)/campione).</li>
	<li>Ruota delicatamente le cellule e incuba per 13 - 30 minuti a RT al buio.</li>
	<li>Aggiungere 500 μL di tampone FACS e filtrare le celle utilizzando provette di polistirene conico per rimuovere le celle aggregate.</li>
	<li>Centrifugare a 400 x g e 4 °C per 3 min.</li>
	<li>Aggiungere 500 μL di tampone legante Annexin V a ciascun tubo e sospendare.</li>
	<li>Analizzare utilizzando un citometro a flusso.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbiota+in+adaptive+immune+homeostasis+and+disease.">The microbiota in adaptive immune homeostasis and disease.</a> Nature. 535 (7610), 75-84 (2016).</li><li>B&#225;rcena, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healthspan+and+lifespan+extension+by+fecal+microbiota+transplantation+into+progeroid+mice.">Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice.</a> Nature Medicine. 25 (8), 1234-1242 (2019).</li><li>Michalovich, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+and+disease+severity+magnify+disturbed+microbiome-immune+interactions+in+asthma+patients.">Obesity and disease severity magnify disturbed microbiome-immune interactions in asthma patients.</a> Nature Communications. 10, 5711 (2019).</li><li>Ansaldo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akkermansia+muciniphila+induces+intestinal+adaptive+immune+responses+during+homeostasis.">Akkermansia muciniphila induces intestinal adaptive immune responses during homeostasis.</a> Science. 364 (6446), 1179-1184 (2019).</li></ol>

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Il microambiente tissutale, comprese le cellule vicine e la matrice extracellulare (ECM), è fondamentale per la generazione dei tessuti e cruciale nel controllo della crescita cellulare e dello sviluppo dei tessuti13. Tuttavia, le colture 2D presentano diversi svantaggi, come l'interruzione delle interazioni cellulari, nonché alterazioni nella morfologia cellulare, ambienti extracellulari e l'approccio della divisione14. I sistemi di coltura cellulare 3D sono stati rigoro...

I probiotici sono i microrganismi più vantaggiosi nell'intestino che migliorano l'omeostasi immunitaria e il metabolismo energetico dell'ospite1. I probiotici di Lactobacillus e Bifidobacterium sono i più avanzati del suo genere trovati nell'intestino2,3. Precedenti indagini hanno dimostrato che Lactobacillus ha effetti inibitori e antiproliferativi su diversi tumori, tra cui il cancro delcolon-retto 4. Inoltre, i probiotici prevengono le malattie infiammatorie intestinali, il morbo di Crohn e la colite ulcerosa5,6. Tuttavia, la maggior parte degli studi con probiotici sono stati eseguiti in monostrati bidimensionali (2D) che vengono coltivati su superfici solide.
I sistemi di coltura artificiale mancano di caratteristiche ambientali, il che non è naturale per le cellule tumorali. Per superare questa limitazione, sono stati sviluppati sistemi di coltura tridimensionali (3D)7,8. Le cellule tumorali in 3D mostrano miglioramenti in termini di meccanismi biologici di base, come la vitalità cellulare, la proliferazione, la morfologia, la comunicazione cellula-cellula, la sensibilità ai farmaci e la rilevanza in vivo9,10. Inoltre, gli sferoidi sono costituiti da tipi multicellulari di cancro del colon-retto e dipendono dalle interazioni cellula-cellula e dalla matrice extracellulare (ECM)11. Il nostro studio precedente ha riportato che il surnatante probiotico privo di cellule (CFS) prodotto utilizzando Lactobacillus fermentum ha mostrato effetti anti-cancro su colture 3D di cellule di cancro del colon-retto (CRC)12. Abbiamo proposto che la CFS sia una strategia alternativa adatta per testare gli effetti probiotici sugli sferoidi 3D12.
Qui, presentiamo un approccio che può ospitare tipi multicellulari di cancro del colon-retto 3D per l'analisi degli effetti terapeutici del surnatante probiotico senza cellule (CFS) su diversi sistemi di mimetismo del cancro del colon-retto 3D. Questo metodo fornisce un mezzo per l'analisi degli effetti probiotici e anti-cancro correlati in vitro.

<table>
	<tbody>
		<tr>
			<td> 
			10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 &#181;l</td>
			<td>Biorad</td>
			<td>4561036</td>
			<td>Pkg of 10</td>
		</tr>
		<tr>
			<td>Applied Biosystems MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>100 covers</td>
		</tr>
		<tr>
			<td>10x transfer buffer</td>
			<td>Intron</td>
			<td>IBS-BT031A</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>10X Tris-Glycine (W/SDS)</td>
			<td>Intron</td>
			<td>IBS-BT014</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case</td>
			<td>Corning</td>
			<td>SCT-200-C</td>
			<td>500 Tubes/Pack, 10 Packs/Case</td>
		</tr>
		<tr>
			<td>BD Difco Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>BD Difco Lactobacilli MRS Broth</td>
			<td>BD</td>
			<td>DF0881-17-5</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell viability assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td>100&#956;l/assay in 96-well plates</td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
			<td>vial of 20 tablets</td>
		</tr>
		<tr>
			<td>Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 &#177; 0.1</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>EMD Millipore Immobilon-P PVDF Transfer Membranes</td>
			<td>fisher Scientific</td>
			<td>IPVH00010</td>
			<td>26.5cm x 3.75m roll; Pore Size: 0.45um</td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>25/Pack, 500/Case</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, US origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit, 25 x 20 &#181;l rxns #1708890</td>
			<td>Biorad</td>
			<td>1708890</td>
			<td>25 x 20 &#181;L rxns</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Biorad</td>
			<td>1725121</td>
			<td>5 x 1 mL</td>
		</tr>
		<tr>
			<td>Lactobacillus fermentum</td>
			<td>Korean Collection for Type Cultures</td>
			<td>KCTC 3112</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C6852-25G</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Methyl Cellulose (3500-5600mPa&#183;s, 2% in Water at 20&#176;C)</td>
			<td>TCI</td>
			<td>M0185</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td>20 plates</td>
		</tr>
		<tr>
			<td>Millex-GS Syringe Filter Unit, 0.22 &#181;m, mixed cellulose esters, 33 mm, ethylene oxide sterilized</td>
			<td>Millipore</td>
			<td>SLGS033SB</td>
			<td>250</td>
		</tr>
		<tr>
			<td>PE Annexin V Apoptosis Detection Kit with 7-AAD</td>
			<td>Biolegend</td>
			<td>640934</td>
			<td>100 tests</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Introgen</td>
			<td>P1304MP</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>89901</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit (250)</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td>250</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Name of Materials/Equipment/Software</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>anti - p-I&#954;B&#945; (B-9)</td>
			<td>Santa cruze</td>
			<td>sc-8404</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-BclxL (H-5)</td>
			<td>Santa cruze</td>
			<td>sc-8392</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-PARP 1 (C2-10)</td>
			<td>Santa cruze</td>
			<td>sc-53643</td>
			<td>50 &#181;l ascites</td>
		</tr>
		<tr>
			<td>anti-&#946;-actin (C4)</td>
			<td>Santa cruze</td>
			<td>sc-47778</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>BD FACSVerse</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>Synergy HTX Multi-Mode Microplate Reader</td>
			<td>BioT</td>
			<td>S1LFA</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo fisher</td>
			<td>HERAcell 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 15 ml</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 50 ml</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile</td>
			<td>Costar</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermofisher</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EnSpire Multimode Reader</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Enspire 2300</td>
		</tr>
		<tr>
			<td>Eppendorf Research Plus Multi Channel Pipette, 8-channel</td>
			<td>Eppendorf</td>
			<td>3122000051</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>TreeStar</td>
			<td></td>
			<td>Ashland, OR, USA</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>115-035-062</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>111-035-144</td>
			<td>2.0 mL</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Inc., San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000 mini</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Incubated shaker</td>
			<td>Lab companion</td>
			<td>SIF-6000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi Gauge Ver. 3.0,</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Optical density (OD)LAMBDA UV/Vis Spectrophotometers</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Phase-contrast microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>SPL microcentrifuge tube 1.5mL</td>
			<td>SPL</td>
			<td>60015</td>
			<td></td>
		</tr>
		<tr>
			<td>SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile</td>
			<td>SPL</td>
			<td>21012</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Vibra-Cell Ultrasonic Liquid Processors</td>
			<td>SONICS-vibra cell</td>
			<td>VC 505</td>
			<td>500 Watt ultrasonic processor</td>
		</tr>
		<tr>
			<td>Vinyl Anaerobic Chamber</td>
			<td>COY LAB PRODUCTS</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating cell death using cell-free supernatant of probiotics in three-dimensional spheroid cultures of colorectal cancer cells

Questo manoscritto descrive un protocollo per valutare le morti delle cellule tumorali in sferoidi tridimensionali (3D) di tipi multicellulari di cellule tumorali utilizzando supernatanti da coltura cellulare di Lactobacillus fermentum , considerati come colture probiotiche. L'uso di colture 3D per testare il surnatante privo di cellule di Lactobacillus (LCFS) è un'opzione migliore rispetto al test in monostrati 2D, soprattutto perché L. fermentum può produrre effetti anti-cancro all'interno dell'intestino. L. fermentum supernatant è stato identificato per possedere un aumento degli effetti anti-proliferativi contro diverse cellule del cancro del colon-retto (CRC) in condizioni di coltura 3D. È interessante notare che questi effetti erano fortemente correlati al modello di coltura, dimostrando la notevole capacità di L. fermentum di indurre la morte delle cellule tumorali. Gli sferoidi stabili sono stati generati da diversi CRC (cellule tumorali del colon-retto) utilizzando il protocollo presentato di seguito. Questo protocollo di generazione di sferoidi 3D consente di risparmiare tempo ed è conveniente. Questo sistema è stato sviluppato per studiare facilmente gli effetti anti-cancro di LCFS in più tipi di sferoidi CRC. Come previsto, gli sferoidi CRC trattati con LCFS hanno fortemente indotto la morte cellulare durante l'esperimento ed espresso specifici marcatori molecolari di apoptosi analizzati mediante qRT-PCR, western blotting e analisi FACS. Pertanto, questo metodo è prezioso per esplorare la vitalità cellulare e valutare l'efficacia dei farmaci anti-cancro.

Descriviamo il protocollo per ottenere sferoidi da diverse linee cellulari di cancro del colon-retto. L'integrazione con metilcellulosa è stata necessaria per generare sferoidi. Presentiamo anche un metodo di preparazione LCFS e presentiamo un modello per studiare la correlazione tra probiotici e cancro del colon-retto. I protocolli di formazione sferoide e di preparazione LCFS sono schematicamente illustrati in Figura 1A,B. Come mostrato nella Figura 2...

Watch this Scientific Journal Video about Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells at JoVE.com

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

Qui vengono presentati i metodi per comprendere gli effetti anti-cancro del surnatante senza cellule di Lactobacillus (LCFS). Le linee cellulari del cancro del colon-retto mostrano decessi cellulari quando trattati con LCFS in colture 3D. Il processo di generazione degli sferoidi può essere ottimizzato a seconda dell'impalcatura e i metodi di analisi presentati sono utili per valutare le vie di segnalazione coinvolte.

Valutazione della morte cellulare utilizzando un surnatante privo di cellule di probiotici in colture sferoidali tridimensionali di cellule tumorali del colon-retto

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

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Cancer Research

5.4K Views.  Korea Research Institute of Standards and Science (KRISS). Qui vengono presentati i metodi per comprendere gli effetti anti-cancro del surnatante senza cellule di Lactobacillus (LCFS). Le linee cellulari del cancro del colon-retto mostrano decessi cellulari quando trattati con LCFS in colture 3D. Il processo di generazione degli sferoidi può essere ottimizzato a seconda dell'impalcatura e i metodi di analisi presentati sono utili per valutare le vie di segnalazione coinvolte.

Video: Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

Utilizzando Genomica Funzionale screening per identificare gli obiettivi farmaco potenzialmente romanzo in Cancer Cell Culture Spheroid

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

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Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Preparazione e dosaggio metabolica della sferoide 3-dimensionale co-colture di cellule di cancro del pancreas e dei fibroblasti

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Lo spostamento verso l'alto: Un semplice e flessibile In Vitro tridimensionale invasione protocollo di analisi

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Matrice a base di tumore umano in un saggio di invasione sferoide tridimensionale

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Valutare la vitalità cellulare e la morte nelle colture di sferoidi 3D delle cellule tumorali

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Isolamento di cellule simili a stelo dalle colture speroidi a 3 dimensioni

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Rilevamento di DNA privo di cellule in campioni di plasma sanguigno di pazienti oncologici

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Un modello tridimensionale di sferoidi per studiare le cellule staminali del cancro del colon

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Modello biomimetico tridimensionale in vitro di neuroblastoma utilizzando scaffold a base di collagene

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

Un modello sferoide tridimensionale per studiare l'interazione tumore-stromale nel carcinoma epatocellulare

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...