Name: a hormone-responsive 3d culture model of the human mammary gland epithelium
Uploaded: 2016-02-07T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

Noi apprezziamo molto i contributi editoriali di Cheryl Schaeberle. Questa ricerca è stata sostenuta da sovvenzioni Avon # 02-2009-093 e 02-2011-095, e NIEHS / NIH ES 08314 di AMS. Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del National Institute of Environmental Health Sciences o il National Institutes of Health.

1. Preparazione dei reagenti <ol><li> Sciogliere il promegestone sintetico progestinico (R5020) e 17-β-estradiolo (E2) in etanolo per fare 10 -3 soluzioni madre M. Sciogliere prolattina in acqua deionizzata distillata per fare un / Soluzione 1 mg ml. Sciogliere il antiestrogeno ICI 182,780 in DMSO per ottenere una soluzione stock 10 -2 M. Conservare queste soluzioni a -20 ° C per un massimo di 6 mesi. </li><li> Carbone destrano (CD) messo a nudo siero e medie CDFBS: <ol><li> Calore inattivare siero bovino fetale (FBS) in un bagno di acqua 57 ° per 30 min. Nota: utilizzare solo vetreria d&#39;acido per il resto di questo protocollo. </li><li> Calcolare la quantità di carbone necessaria. Utilizzando 10% di volume rispetto alla quantità di siero per compensare lo spostamento causato dal carbone, pesare 5% peso / volume carbone attivo in polvere. </li><li> Lavare carbone due volte con acqua distillata deionizzata fredda utilizzando lo stesso volume come la quantità di siero. Per ogni lavaggio centrifuge a 50 g per 2 minuti per far sedimentare il carbone. Rimuovere l&#39;acqua tra un lavaggio. Aggiungere 0,5% peso / volume Destrano T-70 all&#39;ultimo pellet carbone. Risospendere con acqua distillata, acqua deionizzata e centrifugare a 100 xg per 5 min. Aspirare l&#39;acqua. </li><li> Aggiungere il volume appropriato di siero al pellet e risospendere il pellet carbone destrano nel siero. Incubare, durante il rotolamento (6 giri), a 37 ° C per 60 min. Centrifugare a 2000 xg per 20 min. Se surnatante appare torbida, ripetere la centrifugazione; tuttavia, siero non sia completamente chiaro in questo momento, ma verrà eliminato durante la filtrazione. </li><li> Filtrare attraverso un filtro 0,80 micron e poi di nuovo attraverso un filtro da 0,45 micron per rimuovere le particelle più grandi. Infine, filtro sterilizzare con filtro 0.20 micron. Conservare CDFBS in tubi di vetro o T-25 fiasche di coltura filtrato a -20 ° C. </li><li> Carbone Destrano Stripped fetale bovino contenenti supporti (CDFBS media): miscelare 375 ml di fenolo rosso-libero DMEM e 125 ml di DMEM/ F-12. Rimuovere 37,5 ml e sostituirlo con lo stesso volume di carbone Destrano Stripped siero fetale bovino. Aggiungere 10 6 U / ml di penicillina e L-glutammina per 2 mm concentrazione finale. I media risultante è del 75% DMEM, 25% DMEM / F-12, il 7,5% del carbone di legna destrano spogliato-FBS, 2 MM L-glutammina, e 10 6 U / ml di penicillina. </li></ol></li><li> Preparare 10X PBS, 1N NaOH e acqua deionizzata distillata. Preparare sufficiente volume di ciascuna funzione del volume di soluzione di collagene necessario. Sterilizzare tutte le soluzioni di filtraggio. Conservare il 10X PBS e acqua deionizzata sterile distillata a 4 ° C per un massimo di 1 mese. Non conservare la soluzione di NaOH 1N. </li><li> Preparare la soluzione di collagene a 1 mg / ml concentrazione finale secondo il protocollo per la &quot;Procedura gelificazione alternativa per collagene I, coda di topo&quot;, fornito dal produttore. Ogni gel è costituito da 1,5 ml di soluzione di collagene. Ad esempio, preparare 18,5 ml (0,5 ml per tenere conto di perdite durante il pipettaggio) di soluzione di collagene per preparare12 gel. Utilizzare la soluzione di collagene immediatamente o tenere in ghiaccio per un massimo di 2-3 ore. </li><li> Carmine Alum dye: <ol><li> Combina 1 g Carmine e 2,5 g di solfato di potassio e alluminio in 500 ml di acqua distillata. Ebollizione agitazione per 20 min. Guarda con attenzione per evitare bollente. Regolare il volume finale di nuovo a 500 ml con acqua distillata aggiuntivo. </li><li> Aggiungere un cristallo di timolo per l&#39;attività antimicrobica e coprire bottiglia con un foglio di alluminio per proteggerlo dalla luce. Filtrare su un filtro di carta di laboratorio di grado prima di ogni utilizzo. Riutilizzare la soluzione Carmine per un massimo di tre mesi. Conservare a temperatura ambiente, al riparo dalla luce. </li></ol></li><li> soluzione di collagenasi: sciogliere collagenasi in medium / F12 DMEM ad una concentrazione di 0,125% w / v. Non conservare questa soluzione. </li></ol> 2. La cultura 3D di T47D celle a coda di ratto collagene di tipo 1 Gel e trattamento ormonale Nota: tenere pipette sierologiche sterili e punte per pipette a 4 ° C. <ol><li> Prepare una cella singola sospensione ed eseguire una conta cellulare. colture cellulari Split T47D prima di raggiungere il 70% di confluenza. Centrifugare il volume che contiene la quantità di cellule necessarie. Si consideri che un gel dovrebbe contenere circa 75.000 T47D cellule. </li><li> Usando una pipetta a freddo, risospendere il pellet cellulare in volume appropriato di collagene. Si consideri che ciascun gel è costituito da 1,5 ml di collagene. Mescolare le cellule e il collagene delicatamente per evitare l&#39;introduzione di bolle. </li><li> Per impedire l&#39;effetto di elettricità statica sulla distribuzione delle celle, spazzolare i bordi, superiore e inferiore della piastra 12 pozzetti con una spazzola statica di riduzione dopo scartare la piastra. Per evitare la formazione di elettricità statica durante il contatto con la superficie di lavoro della cappa, collocare il piatto sopra l&#39;altro 12-pozzetti (piatto vuoto) che è stato anche spazzolato con il brush.Gently statico-riduzione, versare 1,5 ml della miscela in ogni pozzetto della piastra 12 pozzetti. </li><li> Porre la piastra in un incubatore a 37 ° C per 30 min. rimuovere °e piastra dall&#39;incubatore e rimetterlo nella cappa. </li><li> Utilizzando una punta sterile p200, staccare con cura il gel in un delicato movimento circolare dal bordo del pozzo. Il gel deve anche staccare dal fondo del pozzo. </li><li> Diluire ormoni nei media CDFBS da aggiungere ai pozzetti. Utilizzare E2 e R5020 ad una concentrazione finale di 10 -10 M e prolattina a a 10 -7 M concentrazione finale. Come controllo, utilizzare supporti CDFBS senza ormoni aggiunto, ma ancora contenente il più alto volume di DMSO pari alle scorte ormone utilizzato. Assicurarsi che ogni pozzetto contiene 1,5 ml di media. Collocare le culture 3D all&#39;interno di un 37 ° C, 5% CO 2, 100% umidità tessuto cultura incubatore. Mantenere le culture per 1 o 2 settimane, cambiando i media ogni 2 giorni. </li></ol> 3. Il trattamento del gel per l&#39;intero Monti <ol><li> Rimuovere terreni di coltura e aggiungere 1,5 ml di soluzione salina in ogni pozzetto. </li><li> Trasferire il gel ad una diapositiva. Tagliare il gel a metà con una curvad lama chirurgica. Continuare il trattamento con una metà e salvare l&#39;altra metà per l&#39;analisi istologica. </li><li> Trasferire la metà di gel per tutto il monte a una diapositiva. Lasciare asciugare il gel per 5 minuti e poi trasferire la diapositiva di una vaschetta Coplin contenente il 10% di formalina. Nota: tutte le fasi di incubazione dovrebbe essere fatto a temperatura ambiente. </li><li> Fissare gel in una vaschetta Coplin su un agitatore O / N. </li><li> Lavare i vetrini due volte con PBS per 10 minuti ciascuno su agitatore. Trasferire i vetrini al 70% EtOH; incubare per 1 ora su agitatore. scivoli Trasferire Carmine Alum O / N. </li><li> Il giorno successivo, disidratano scorre su shaker in 70% EtOH, 95% EtOH e 100% EtOH per 1 ora ciascuno. Trasferire i vetrini a xilene due volte per 1 ora ogni e continuare a shaker. Utilizzare a base di toluene mezzo di montaggio per montare i gel e 1,5 mm di spessore coprioggetto. </li><li> Esaminare i supporti interi in condizioni di luce normale (microscopio o stereoscopio). Per un&#39;analisi più dettagliata della forma e lume di utilizzare la microscopia confocale e visualizzare Carmine tintura con HeNe 633 nm laser. </li></ol>4. Il trattamento gel per l&#39;elaborazione di analisi Istologia <ol><li> Trasferire la restante metà del gel di un flaconcino di vetro da 20 ml contenente 10% di formalina e fissare sulla agitatore O / N. </li><li> Lavare gel due volte con PBS per 10 minuti ciascuno su agitatore. Luogo gel in cassette di elaborazione per paraffina. </li><li> gel Trasferire il 70% EtOH, incubare per 1 ora e seguono con il 95% EtOH per 1 ora, cambiano al fresco 95% EtOH per 1 ora, 100% EtOH per 1 ora, e cambiare al fresco 100% EtOH per 1 ora. A questo punto, i gel possono essere conservati in 100% EtOH O / N a RT o incubate con xilene per 1 ora e poi di nuovo in xilene fresco per 1 ora. </li><li> cassette di trasferimento di paraffina per 1 ora a 60 ° C in incubatore vuoto, ripetere con paraffina fresco due volte. </li><li> Riempire stampi in plastica con paraffina pulito. Aprire il cassetto, rimuovere il gel e metterlo nello stampo contenente paraffina plastica. Riempire con paraffina e aggiungere superiore del cassetto contenente l&#39;etichetta sulla parte superiore e lasciare blocco freddo. </li><li> Rimuovere il plastmuffa ic e la sezione del blocco utilizzando un microtomo. Ottenere 5 um sezioni spesse per HE colorazione e immunocitochimica. </li></ol> 5. estrazione di cellule da cellule dal gel utilizzando un trattamento Collagenasi <ol><li> Rimuovere il gel dal pozzo e trasferirlo ad una piastra di Petri contenente PBS. brevemente gel di risciacquo. </li><li> Trasferire il gel ad una provetta da 15 ml contenente 2 ml di 0,125% collagenasi. Pipettare su e giù 4-5 volte per rompere il gel in piccoli pezzi. </li><li> Incubare a 37 ° C per 25 min (fino gel si è sciolto e cellule sono in sospensione) </li><li> Aggiungere 2 ml di siero integrato terreno di coltura. Raccogliere le cellule per centrifugazione (1.000 xg, 5 min), il surnatante scarti, risospendere le cellule e contare. </li></ol>

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	<li>Barnes, 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=From+single+cells+to+tissues:+interactions+between+the+matrix+and+human+breast+cells+in+real+time.">From single cells to tissues: interactions between the matrix and human breast cells in real time.</a> PLoS ONE. 9, e93325 (2014).</li><li>Berthois, Y., Katzenellenbogen, J. A., Katzenellenbogen, B. 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M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+collagen+reorganization+on+mammary+epithelial+morphogenesis+in+a+3D+culture+model.">The role of collagen reorganization on mammary epithelial morphogenesis in a 3D culture model.</a> Biomaterials. 31, 3622-3630 (2010).</li><li>Dhimolea, E., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+epithelial+tissue+morphology+is+affected+in+3D+cultures+by+species-specific+collagen-based+extracellular+matrix.">Breast epithelial tissue morphology is affected in 3D cultures by species-specific collagen-based extracellular matrix.</a> J.Biomed.Mat.Res.A. 100, 2905-2912 (2012).</li><li>Krause, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+regulation+of+breast+tubulogenesis+using+extracellular+matrix+composition+and+stromal+cells.">Dual regulation of breast tubulogenesis using extracellular matrix composition and stromal cells.</a> Tissue Eng Part A. 18, 520-532 (2012).</li><li>Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+in+vitro.+culture+model+to+study+stromal-epithelial+interactions+in+the+mammary+gland.">A novel 3D in vitro. culture model to study stromal-epithelial interactions in the mammary gland.</a> Tissue Eng.Part C Methods. 14, 261-271 (2008).</li><li>Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microenvironment+determines+the+breast+cancer+cells'+phenotype:+organization+of+MCF7+cells+in+3D+cultures.">The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures.</a> BMC Cancer. 10, 263-275 (2010).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat.Methods. 4, 359-365 (2007).</li><li>Martinson, H. A., Jindal, S., Durand-Rougely, C., Borges, V. F., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing-like+immune+program+facilitates+postpartum+mammary+gland+involution+and+tumor+progression.">Wound healing-like immune program facilitates postpartum mammary gland involution and tumor progression.</a> Int.J.Cancer. , (2014).</li><li>Soto, A. M., Murai, J. T., Siiteri, P. K., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+proliferation:+evidence+for+negative+control+on+estrogen-sensitive+T47D+human+breast+cancer+cells.">Control of cell proliferation: evidence for negative control on estrogen-sensitive T47D human breast cancer cells.</a> Cancer Res. 46, 2271-2275 (1986).</li><li>Speroni, L., 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=Hormonal+regulation+of+epithelial+organization+in+a+3D+breast+tissue+culture+model.">Hormonal regulation of epithelial organization in a 3D breast tissue culture model.</a> Tissue Eng.Part C Methods. 20, 42-51 (2014).</li><li>Vignon, F., Bardon, S., Chalbos, D., Rochefort, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiestrogenic+effect+of+R5020,+a+synthetic+progestin+in+human+breast+cancer+cells+in+culture.">Antiestrogenic effect of R5020, a synthetic progestin in human breast cancer cells in culture.</a> J.Clin.Endocrinol.Metab. 56, 1124-1130 (1983).</li></ol>

The authors have nothing to disclose.

Here, we describe a hormone-sensitive 3D culture model to test the action of hormones on breast epithelium. The response to hormones can be assessed at the tissue morphology, cell proliferation and gene expression levels 12. One limitation of this technique is that visualization during the culture period is restricted to light microscopy since the cultures are grown in a plastic bottom plate. The 3D culture system could be adapted to glass bottom plates to allow for live imaging of the cultures 1.
The way in which cells are maintained appears to be critical for retention of hormone-responsive phenotypes. It is important to frequently check for E2 responsiveness in these cells. We recommend performing an E2 dose-response curve on the fourth passage after thawing a new vial, and routinely after every 10 passages thereafter. An E2 concentration of 10 -10 M will typically yield three times the control cell number in 2D culture and two times in 3D culture when compared to those grown in CDFBS medium for 1 week 12. Phenol red-free media should be used for all experiments involving hormone-responsiveness, due to the proven estrogenicity of phenol red 2. In addition, the brands listed in this protocol are current without hormonal activity. If the estrogenic response in 2D or 3D changes, the plastic materials used for tissue culture should be evaluated for leaching of hormonally-active compounds. 
The presence of static electricity during gel preparation may affect cell distribution. This may cause the cells to quickly sink to the bottom of the well during congealing and then grow as a 2D monolayer. In this case, the gel appears mostly empty of cells with them attached directly to the bottom plastic surface. To prevent this effect, it is recommended to brush the borders, top and bottom of the 12-well plate with a static-reducing brush after unwrapping the plate. To avoid static charge buildup during contact with the hood working surface, place this plate on top of another 12-well plate (blank plate) that has also been brushed with the static-reducing brush. These recommendations are especially useful during periods of low ambient humidity. 
As opposed to existing "on top" culture models9, the culture system that we describe here allows for the epithelial cells to form 3D structures which are embedded in a matrix. Furthermore, this culture system enables for the study of the influence of extracellular matrix components, such as collagen fibers, and mechanical properties, such as matrix stiffness, in hormone-instructed morphogenesis of 3D epithelial structures 1,12. 
The hormone-sensitive 3D culture model described herein also allows for the addition of cell types considered of relevance in breast tissue morphogenesis, such as fibroblasts, adipocytes and circulating blood cells 6,8,10. Finally, testing of hormonal influence on the breast epithelium in this 3D culture model is not limited to endogenous hormones alone. The response to environmental estrogenic compounds and other hormonally-active chemicals can also be tested to uncover their effects and those elicited when tested in the presence of the natural, endogenous steroidal and polypeptidic hormones.

Diversamente culture standard 2D, modelli surrogati di coltura cellulare 3D permettono lo studio del comportamento delle cellule epiteliali in un contesto fisiologicamente rilevanti, uno simile a un tessuto. culture 3D della ghiandola mammaria hanno contribuito a chiarire molti aspetti dello sviluppo della ghiandola mammaria e neoplasie. Tuttavia, la maggior parte dei modelli di coltura 3D attualmente disponibili non sono adatti per studiare l&#39;azione ormonale perché le linee di cellule epiteliali umane utilizzate per l&#39;attività carenti dell&#39;ormone del recettore espressione 6,7,9. Qui, descriviamo un modello di coltura 3D dell&#39;epitelio mammario umano che è adatto per studiare l&#39;azione dell&#39;ormone 12. Questo modello utilizza la linea commercialmente disponibile ormono-sensibile umano epiteliali seno cellule, T47D 3,11,13, originariamente derivato da un versamento pleurico ottenuto da 54 anni paziente con un carcinoma duttale infiltrante della mammella. Usiamo ratto collagene coda di tipo 1 come matrice. Questo culto 3DModello ure è appropriato per lo studio dell&#39;azione dei tre principali ormoni mammotropic (estradiolo, promegestone (un analogo di progesterone), e prolattina) sulle cellule epiteliali del seno umane. Ormone-indotta morfologia epiteliale può essere valutata quantitativamente nel tempo da analisi morfometrica 12. Una densità di semina adeguato permette a queste culture 3D per essere conservati per 2 settimane. A questo punto, lo sviluppo di strutture è sufficiente per un robusto valutazione quantitativa dell&#39;azione ormonale sulla morfologia epiteliale. I gel possono essere raccolte in momenti precedenti per la proliferazione cellulare e le analisi di espressione genica. Inoltre, questo modello è adatto per testare gli effetti di un trattamento ormonale sequenziale; per esempio, dopo il trattamento con estradiolo durante la prima settimana e la sostituzione con altro ormone / combinazione di ormoni durante la settimana seguente. L&#39;effetto dei composti estrogenici e antiestrogeni, come ICI 182,780, può anche essere Stuè morto con questa cultura modello 3D 12.

<table><tbody><tr><td>12-well Tissue Culture Plates (Falcon)</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>15 ml polystyrene conical Tubes</td><td>Fisher Scientific</td><td>14-959-49D</td><td></td></tr><tr><td>Activated Charcoal</td><td>Sigma</td><td>C-5510</td><td></td></tr><tr><td>Carmine Alum</td><td>Sigma</td><td>C1022-100G</td><td></td></tr><tr><td>Collagenase, Type 3</td><td>Worthington</td><td>S0C11784</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM510</td><td>Equiped with HeNe 633nm laser</td></tr><tr><td>Dextran T-70</td><td>Abersham/Pharmacia</td><td>17-0280-01</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red&amp;nbsp;</td><td>Life Technologies</td><td>11339-021</td><td>Phenol red-free media for hormone use</td></tr><tr><td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td><td>Life Technologies</td><td>11054-020</td><td>Phenol red-free media for hormone use</td></tr><tr><td>17-&amp;beta;-Estradiol</td><td>EMD Millipore</td><td>3301</td><td>Dissolved in Ethanol</td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30070.03</td><td>For use with hormones, must be Charcoal Dextran stripped&amp;nbsp;</td></tr><tr><td>Filters (115 ml)</td><td>Nalgene</td><td>380-0080, 245-0045, 120-0020</td><td>0.88, 0.45, 0.20 micron, respectively&amp;nbsp;</td></tr><tr><td>Formalin, 10%</td><td>Fisher Scientific</td><td>SF93-20</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>ICI 182,780 (fulvestrant)</td><td>Sigma Aldrich</td><td>I4409-25MG</td><td>Dissolved in DMSO</td></tr><tr><td>Microtome&amp;nbsp;</td><td>Leica</td><td>RM2155</td><td></td></tr><tr><td>Tissue embedding media</td><td>McCormick Scientific</td><td>39502004</td><td></td></tr><tr><td>Penicillin</td><td>Sigma</td><td>7794-10MU</td><td>Dissolved in 10 ml of distilled deionized water</td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td></td></tr><tr><td>Prolactin</td><td>Sigma Aldrich</td><td>L4021-50UG</td><td>Dissolved in distilled deionized water</td></tr><tr><td>Promegestone</td><td>Perkin Elmer</td><td>NLP004005MG</td><td>Dissolved in Ethanol</td></tr><tr><td>Rat-Tail Collagen&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>354236</td><td>Lots may contain varying concentrations, note accordingly</td></tr><tr><td>Scalpel</td><td>Miltex</td><td>4311</td><td></td></tr><tr><td>Semi-enclosed Benchtop Tissue Processor</td><td>Leica</td><td>TP1020</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Prepare 1N NaOH stock</td></tr><tr><td>StaticMaster Anti-static brush&amp;nbsp;</td><td>Amstat</td><td>C3500</td><td></td></tr><tr><td>Stripette Serological Pipettes&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>4101</td><td></td></tr><tr><td>T-25 flasks</td><td>Corning&amp;nbsp;</td><td>430168</td><td></td></tr><tr><td>Tissue Cassettes</td><td>Fisher Scientific</td><td>15-200-403E</td><td></td></tr><tr><td>Wheaton Vials, Glass, 20 ml</td><td>Fisher Scientific</td><td>03-341-25D&amp;nbsp;</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>VWR</td><td>95057-822</td><td></td></tr></tbody></table>

a hormone-responsive 3d culture model of the human mammary gland epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

La Figura 1 riassume il procedimento per la preparazione delle culture 3D ormone-sensibile. strutture epiteliali sono osservati in interi monti del gel in coltura per 2 settimane in presenza di E2 da solo e in combinazione con altri ormoni. Solo le cellule singole o gruppi di 2-3 cellule sono presenti quando ormoni vengono aggiunti al mezzo di coltura (media CDFBS) (Figura 2). Questa condizione serve come controllo negativo. Le cellule in strutture di forma cultura 3D che variano per forma, dimensione e presenza lumen. La distribuzione delle strutture nel gel è tipicamente eterogenea. Come illustrato in precedenza, ci sono generalmente più strutture lungo i confini del gel di quanto visto al centro 4. A causa di questa eterogeneità spaziale, confronti tra i campioni devono essere limitati alle regioni corrispondenti tra i campioni. Le specie da cui collagene di tipo 1 è derivato influenza il fenotipo di unnd qualità delle risultanti strutture epiteliali 5. Rat coda collagene di tipo 1, come qui utilizzato, è stato scelto poiché semina in bovini collagene di tipo 1 provocato formazione di gruppi di cellule disordinati (Figura 3). Un&#39;analisi dettagliata delle strutture epiteliali può essere raggiunto utilizzando la microscopia confocale (Figura 4). Risultati del trattamento E2 in strutture arrotondate e allungate (Figura 4A). La presenza di E2 e risultati prolattina in strutture germoglianti più (Figura 4B), mentre la presenza di E2 e risultati promegestone in strutture irregolari con proiezioni cellulari ricordano lato ramificazione (Figura 4C). solo E2 ed E2, più prolattina addensare strutture dei tessuti se misurato dalla crescita nella direzione z, e rispetto a quelli osservati nella E2 più promegestone. Al fine di quantificare il numero di cellulare dopo un trattamento ormonale, le cellule sono extracted dal gel mediante trattamento collagenasi. Ad esempio, con questo metodo si può studiare come l&#39;antiestrogeno ICI 182,780 inibisce l&#39;effetto di E2 sulla proliferazione delle cellule nelle colture 3D (Figura 5). <img alt="Figura 1" src="/files/ftp_upload/53098/53098fig1.jpg" /> Figura 1. Procedimento per la preparazione di colture 3D ormone-sensibile. Brevemente, una cella singola sospensione di cellule T47D è mescolato con coda di ratto collagene di tipo 1 e versata in pozzetti di una piastra 12 pozzetti. I gel possono congelare per 30 min a 37 ° C / 5% CO2 incubatore. Terreno di coltura viene aggiunto ei gel sono staccato dal pozzo. Le culture possono essere raccolte dopo 1 o 2 settimane a seconda del punto finale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig1large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> . Figura 2. monti interi di culture 3D dopo colorazione con colorante Carmine Allume (A) tutto il monte di mezzo gel colorato con Carmine allume per visualizzare le strutture epiteliali, un esempio di ROI per l&#39;analisi della morfologia è indicato da una scatola; WM di culture 3D coltivato per 2 settimane in (B) CDFBS Media (senza ormoni aggiunti) e (C) CDFBS multimediale integrato con E2 e prolattina. Barre di scala sono 5.000 micron (A) e 500 micron (B &amp; C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig2large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53098/53098fig3.jpg" /> Figura 3. Confronto delle matrici utilizzate nella cultura 3D. (A) BoviNE collagene di tipo 1 risultati in gruppi disorganizzati di cellule, in contrasto con le strutture organizzate (B) osservato quando si utilizza coda di topo collagene di tipo bar 1. Scala:. 100 micron <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53098/53098fig4.jpg" /> Figura 4. immagini confocali strutture T47D osservata in 3-D gel di collagene dopo 2 settimane. Il trattamento con (A) E2, (B) + E2 prolattina e (C) E2 + R5020. Le frecce indicano le proiezioni citoplasmatici. Spessore delle strutture epiteliali era 24, 40 e 20 micron rispettivamente. Barra di scala:. 40 micron <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig4large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Figura 5. culture 3D trattati con sola E2 o E2 più ICI 182,780 per 1 settimana. Conta delle cellule dopo l&#39;estrazione delle cellule utilizzando un trattamento collagenasi. (A) CDFBS media, (B) CDFBS medio più E2 a 1x10 -10 M, (C) ICI 182,780 a 1x10 -8 M plus E2 a 1x10 -10 M e (D) ICI 182,780 a 1x 10-7 M plus E2 a 1x10 -10 M. Bar:. SEM <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig5large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Una cultura Modello 3D Ormone-sensibile della Human ghiandola mammaria Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

a-hormone-responsive-3d-culture-model-human-mammary-gland

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.7K Views.  Tufts University School of Medicine. We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Video: A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Differenziazione delle cellule epiteliali del seno del topo HC11 e EpH4

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Ricerca

Ricerca sul cancro

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 ...

Medicina

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Trasformazione indotta dal mediatore dei fosfolipidi in colture tridimensionali

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

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 ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Generazione e imaging di organoidi epiteliali di topo e umani da tessuto mammario normale e tumorale senza passaggio

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Tessuti epiteliali Ingegneria tridimensionali integrati all&#39;interno di matrice extracellulare

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Bioingegneria

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Generazione di organoidi mammari mosaici per provepsinizzazione differenziale

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Biologia dello sviluppo

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

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

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

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

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

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

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento di cellule epiteliali mammarie da tridimensionale Mixed-cell Spheroid Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Una cultura Modello 3D Ormone-sensibile della Human ghiandola mammaria Epithelium

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