Name: a hormone-responsive 3d culture model of the human mammary gland epithelium
Uploaded: 2016-02-07T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

Wir freuen uns über die redaktionellen Beiträge von Cheryl Schaeberle. Diese Forschung wurde unterstützt von Avon Grants # 02-2009-093 und 02-2011-095, und NIEHS / NIH ES 08314 zu AMS. Der Inhalt ist allein in der Verantwortung der Autoren und stellen nicht notwendigerweise die offizielle Meinung des National Institute of Environmental Health Sciences oder der National Institutes of Health vertreten.

1. Vorbereitung der Reagenzien <ol><li> Man löst das synthetische Gestagen Promegeston (R5020) und 17-β-Estradiol (E2) in Ethanol zu machen 10 -3 M Stammlösungen. Löse Prolaktin in destilliertem deionisiertem Wasser auf eine 1 mg / ml Stammlösung machen. Man löst das Antiöstrogen ICI 182,780 in DMSO auf 10 -2 M Stammlösung machen. Bewahren Sie diese Lösungen bei -20 ° C bis zu 6 Monaten. </li><li> Holzkohle-Dextran (CD) gereinigten Serum und CDFBS Medium: <ol><li> Wärme-Inaktivierung von fötalem Rinderserum (FBS) in einem 57 ° C Wasserbad für 30 min. Hinweis: Verwenden Sie nur säuregewaschenen Glaswaren für den Rest dieses Protokolls. </li><li> Berechnen der Menge an Aktivkohle benötigt. Verwendung von 10% größeres Volumen als die Menge an Serum für die Verschiebung von der Holzkohle, wiegen 5% wt / vol Aktivkohlepulver zu kompensieren. </li><li> Waschen Kohle zweimal mit kaltem entionisiertem destilliertem Wasser das gleiche Volumen wie die Menge des Serums. Für jede Wasch centrifuge bei 50 g für 2 min die Holzkohle zu pelletieren. Entfernen Sie das Wasser zwischen den Waschgängen. In 0,5% w / v Dextran T-70 bis zum letzten Kohle Pellet. Resuspendieren mit destilliertem, entionisiertem Wasser und Zentrifuge bei 100 × g für 5 min. Saugen Sie das Wasser. </li><li> Fügen Sie den entsprechenden Volumen von Serum zu dem Pellet und erneut zu suspendieren die Holzkohle-Dextran-Pellet in das Serum. Man inkubiert, während (6 RPM) Walzen, bei 37 ° C für 60 min. Zentrifuge bei 2.000 × g für 20 min. Wenn überstehende trübe erscheint, wiederholen Zentrifugation; jedoch Serum kann nicht völlig klar sein, zu diesem Zeitpunkt aber wird während der Filtration geklärt werden. </li><li> Man filtriert durch ein 0,80-Mikron-Filter und dann erneut durch ein 0,45 Mikrometer-Filter, um größere Partikel zu entfernen. Schließlich Filter-Sterilisieren mit einem 0,20-Mikron-Filter. Shop gefiltert CDFBS in Glasröhrchen oder T-25 Kulturflaschen bei -20 ºC. </li><li> Charcoal Dextran Stripped Fetal Bovine haltigen Medien (CDFBS Medien): mix 375 ml Phenolrot-freiem DMEM und 125 ml DMEM/ F-12. Entfernen 37,5 ml und ersetzen Sie es mit dem gleichen Volumen von Holzkohle Dextran Stripped Fetal Bovine Serum. Fügen Sie 10 6 U / ml Penicillin und L-Glutamin in eine 2 mM Endkonzentration. Das resultierende Medium beträgt 75% DMEM, 25% DMEM / F-12, 7,5% Charcoal Dextran abgestreift-FBS, 2 mM L-Glutamin und 10 &amp; sup6; U / ml Penicillin. </li></ol></li><li> Bereiten 10X PBS, 1 N NaOH und destilliertem deionisiertem Wasser. Bereiten genügend Volumen von jedem nach dem Volumen der Kollagenlösung benötigt. Sterilisieren Sie alle Lösungen durch Filterung. Bewahren Sie die 10x PBS und sterilem destilliertem entionisiertem Wasser bei 4 ° C für bis zu 1 Monat. Bewahren Sie das 1 N NaOH-Lösung. </li><li> Bereiten Kollagenlösung bei einer 1 mg / ml Endkonzentration nach dem Protokoll für &quot;Alternate Gelierung Verfahren für Kollagen I, Rat tail&quot; durch den Hersteller zur Verfügung gestellt. Jedes Gel wird von 1,5 ml Kollagenlösung hergestellt. Zum Beispiel, bereiten 18,5 ml Kollagenlösung (0,5 ml für den Verlust beim Pipettieren zu berücksichtigen) vorbereiten12 Gele. Verwenden Sie die Kollagenlösung sofort oder halten auf dem Eis für bis zu 2-3 Stunden. </li><li> Carmine Alum Farbstoff: <ol><li> Kombinieren Sie 1 g Carmine und 2,5 g Aluminiumkaliumsulfat in 500 ml destilliertem Wasser. Kochen während 20 Minuten unter Rühren. Achten Sie sorgfältig Überkochen zu vermeiden. Passen endgültige Volumen wieder auf 500 ml mit zusätzlichem destilliertem Wasser. </li><li> Fügen Sie einen Kristall von Thymol auf antimikrobielle Aktivität und decken Flasche mit Aluminiumfolie vor Licht zu schützen. Man filtriert durch ein Laborqualität Papierfilter vor jedem Gebrauch. Wiederverwendung der Carminlösung für bis zu drei Monaten. Aufbewahren bei Raumtemperatur, vor Licht geschützt. </li></ol></li><li> Kollagenase-Lösung: Man löst die Kollagenase in DMEM / F12-Medium in einer Konzentration von 0,125% w / v. Sie diese Lösung nicht speichern. </li></ol> 2. 3D Culture von T47D Zellen in Rattenschwanz Kollagen Typ 1 Gels und Hormonbehandlung Hinweis: Halten Sie sterilen serologischen Pipetten und Pipettenspitzen bei 4 ° C. <ol><li> Prepaerneut eine Einzelzellsuspension und führen Sie eine Zellzahl. Split T47D Zellkulturen, bevor sie erreichen 70% Zusammenfluss. Zentrifugieren Sie die Lautstärke, die die Menge von Zellen benötigt enthält. Bedenken Sie, dass ein Gel sollte etwa 75.000 T47D Zellen enthalten. </li><li> Verwendung eines kalt Pipette resuspendieren das Zellpellet in dem entsprechenden Volumen von Kollagen. Bedenken Sie, dass jeder Gel von 1,5 ml Kollagen aus. Mischen Sie die Zellen und das Kollagen sanft zu vermeiden Blasen einzuführen. </li><li> Um die Wirkung von statischer Elektrizität auf Zellverteilung verhindern, Pinsel die Ränder, oben und unten an der 12-Well-Platte mit einer statischen reduzierenden Bürste nach den Teller auspacken. Um zu vermeiden, statische Aufladung bei Berührung mit der Arbeitsfläche der Haube, legen Sie diese Platte auf eine andere 12-Well-Platte (Blindplatte), die auch mit der statischen mindernde brush.Gently gebürstet wurde, gießen 1,5 ml der Mischung in jede Vertiefung der 12-Well-Platte. </li><li> Die Platte wird in einem Brutschrank bei 37 ° C für 30 Minuten. entfernen the Platte aus dem Inkubator und legen Sie sie in die Kapuze zurück. </li><li> Mit einer p200 sterile Spitze, nehmen sorgfältig das Gel in einer sanften kreisenden Bewegungen von der Grenze des Brunnens. Das Gel sollte auch aus dem Boden des Bohrlochs zu lösen. </li><li> Verdünnte Hormone in CDFBS Medien zu den Vertiefungen hinzugefügt werden. Verwenden E2 und R5020 bei einer 10 -10 M Endkonzentration und Prolaktin auf eine bei 10 -7 M Endkonzentration. Als Kontrolle verwenden CDFBS Medien ohne Hormone zugesetzt, aber immer noch das höchste Volumen von DMSO in Höhe der Hormon Lagerbestände enthält. Stellen Sie sicher, dass jede Vertiefung 1,5 ml Medium enthält. Platzieren Sie die 3D-Kulturen in einem 37 ° C, 5% CO 2, 100% Feuchtigkeit Gewebekultur-Inkubator. Pflegen Sie die Kulturen für ein oder 2 Wochen die Medien alle 2 Tage zu ändern. </li></ol> 3 Gel-Verarbeitung für Whole Mounts <ol><li> Entfernen Kulturmedien und fügen Sie in jede Vertiefung 1,5 ml PBS. </li><li> Übertragen Sie das Gel auf einen Objektträger. Schneiden Sie das Gel in ein halbes Kurved chirurgischen Klinge. Weiter Verarbeitung mit der Hälfte und die andere Hälfte zur histologische Analyse speichern. </li><li> Übertragen Sie die Hälfte Gel für ganze auf einen Objektträger montieren. Lassen Sie Gel trocken für 5 Minuten und dann übertragen Sie die Folie in eine Coplin-Gefäß 10% Formalin enthält. Anmerkung: Alle Inkubationen sollte bei RT geschehen. </li><li> Fix Gele in einem Coplin-Gefäß auf einem Schüttler O / N. </li><li> Wash gleitet zweimal mit PBS für jeweils 10 Minuten auf einem Schüttler. Objektträger nach 70% EtOH; 1 h auf einem Schüttler inkubiert. Objektträger zu Carmine Alum O / N. </li><li> Am nächsten Tag entwässern gleitet auf Schüttler in 70% EtOH, 95% EtOH und 100% EtOH für 1 Stunde jede. Objektträger zweimal 1 Stunde jeden und halten am Schüttler zu Xylol. Verwenden Toluol-basierte Montage Medium der Gele und 1,5 mm dicken Deckgläser zu montieren. </li><li> Untersuchen ganze Halterungen unter normalen Licht (Mikroskop oder Stereoskop). Für eine genauere Analyse der Form und Lumen nutzen konfokale Mikroskopie und Carmine Farbstoff mit He-Ne-633-nm-Laser visualisieren. </li></ol>4. Gel Verfahren für die Verarbeitung für die Histologie Analyse <ol><li> Die restliche Hälfte des Gels auf eine 20 ml-Glasfläschchen, das 10% Formalin und legen Sie auf den Schüttler O / N. </li><li> Waschen Sie Gele zweimal mit PBS für jeweils 10 Minuten auf einem Schüttler. Platz Gele in Verarbeitung Kassetten für Paraffineinbettung. </li><li> Transfer Gele bis 70% EtOH, Inkubation für 1 Stunde und folgen mit 95% EtOH für 1 Stunde, ändern an die frische 95% EtOH für 1 h, 100% EtOH für 1 Stunde, und wechseln Sie in frischem 100% EtOH für 1 Stunde. An diesem Punkt, Gele können für 1 Stunde und dann wieder in frischem Xylol für 1 Stunde in 100% EtOH O / N bei RT oder inkubiert mit Xylol gespeichert werden. </li><li> Transfer Kassetten für 1 h bei 60 ° C im Vakuum Inkubator wiederholt mit frischem Paraffin zweimal mehr Paraffin. </li><li> Füllen Kunststoffformen mit sauberem Paraffin. Öffnen Sie die Kassette entfernen Sie das Gel und legen Sie sie in der Kunststoffform, die Paraffin. Füllen Sie mit Paraffin up und top In der Kassette mit dem Etikett nach oben enthalten, und lassen Block cool. </li><li> Entfernen Sie die plastic Form und Abschnitt der Block mit einem Mikrotom. Erhalten Sie 5 um dicke Abschnitte für HE-Färbung und Immunzytochemie. </li></ol> 5. Gewinnung von Zellen aus Zellen aus Gele Collagenase-Behandlung <ol><li> Entfernen Gel aus dem Brunnen und überträgt es auf eine Petrischale mit PBS. Spülen Gel kurz. </li><li> Übertragen Sie das Gel in ein 15 ml Röhrchen mit 2 ml 0,125% Collagenase. Pipette auf und ab 4-5 mal, um das Gel in kleine Stücke zu brechen. </li><li> bei 37 ° C inkubieren für 25 min (bis-Gel aufgelöst und die Zellen werden in Suspension) </li><li> 2 ml Serum ergänzten Kulturmedium. Sammeln Sie die Zellen durch Zentrifugation (1000 × g, 5 min), Überstand verwerfen, resuspendieren Zellen und zählen. </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=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.</...

Im Gegensatz zu Standard-2D-Kulturen erlauben Surrogat 3D-Zellkulturmodelle für die Untersuchung von epithelialen Zellverhalten in einem physiologisch relevanten Kontext man eine Gewebe ähnelt. 3D-Kulturen der Brustdrüse viele Aspekte der Brustdrüse Entwicklung und Neoplasie aufzuklären haben dazu beigetragen. Die Mehrzahl der 3D-Kulturmodellen jedoch derzeit verfügbar sind ungeeignet Hormonwirkung zu studieren, weil die menschlichen epithelialen Zelllinien für die Aufgabe eingesetzt 6,7,9 fehlt Hormonrezeptorexpression. Hier beschreiben wir eine 3D-Kultur-Modell der menschlichen Brust-Epithel, die geeignet ist, um die Hormonwirkung 12 studieren. Dieses Modell verwendet die im Handel erhältlich Hormone ansprechenden menschlichen Brust epithelialen Zelllinie T47D 3,11,13, die ursprünglich von einem Pleuraerguss von einer 54-jährigen Patientin mit einer Infiltrations duktale Mammakarzinom erhalten abgeleitet wurden. Wir verwenden Rattenschwanz Kollagen vom Typ 1 als Matrix. Diese 3D-Kulture-Modell ist für die Untersuchung der Wirkung von den drei Haupt mammotropic Hormone (Östradiol, Promegeston (ein Analogon von Progesteron) und Prolaktin) angemessen auf die menschliche Brust Epithelzellen. Hormonbedingten können epitheliale Morphologie quantitativ im Laufe der Zeit durch morphometrische Analyse 12 bewertet werden. Eine geeignete Aussaatdichte ermöglicht diese 3D-Kulturen für 2 Wochen aufbewahrt werden. Zu dieser Zeit ist die Entwicklung von Strukturen für eine ausreichend robust quantitative Bewertung der Hormonwirkung auf epitheliale Morphologie. Gele können auch zu früheren Zeitpunkten für die Zellproliferation und die Genexpression analysiert geerntet werden. Darüber hinaus ist dieses Modell die Auswirkungen einer sequentiellen Hormonbehandlung zu testen geeignet; zum Beispiel nach der Behandlung mit Estradiol während der ersten Woche und Austausch mit anderen Hormon / Kombination von Hormonen während der folgenden Woche. Die Wirkung des Östrogen-Verbindungen und Antiöstrogene, wie ICI 182,780, können auch stu werdenstarb dieses 3D-Kultur-Modell 12 mit.

<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.

Abbildung 1 fasst die Vorgehensweise für die hormonsensitive 3D-Kulturen vor. Epithelialen Strukturen werden in ganze Halterungen von Gelen kultiviert für 2 Wochen in Gegenwart von E2 allein und in Kombination mit anderen Hormonen beobachtet. Nur einzelne Zellen oder Gruppen von 2-3 Zellen vorhanden sind, wenn keine Hormone zu dem Kulturmedium (CDFBS mittel) (Bild 2) zugesetzt werden. Dieser Zustand dient als Negativkontrolle. Die Zellen in 3D-Kultur bilde...

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.

Ein Hormon ansprechenden 3D-Kultur Modell des menschlichen Brustdrüse Epithel

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

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Research

JoVE Journal

Bioengineering

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

Serie: 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.

Differenzierung von Mausbrustepithel-HC11- und EpH4-Zellen

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 ...

Forschung

Krebsforschung

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.

Eine dreidimensionale Zellkulturmodell zu primären humanen Knochenmark und seine Malignome Studieren

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

Medizin

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.

Phospholipid-Mediator-induzierte Transformation in dreidimensionalen Kulturen

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.

Dreidimensional Kulturen: Ein Werkzeug, um Architektur zu studieren Normale acinar gegen maligne Transformation von Brustzellen

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.

Quantifizierung der Brustkrebs-Zelle Invasivität Mit einem Drei-dimensionale (3D) Modell

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.

Erzeugung und Bildgebung von Epithelorganoiden von Mäusen und Menschen aus normalem und Tumor-Brustgewebe ohne Passaging

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.

Technik Dreidimensionale Epithelgewebe Eingebettet in extrazellulären Matrix

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

Biotechnik

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.

Generierung von Mosaik-Mammary-Organoiden durch Differential-Trypsinisierung

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

Entwicklungsbiologie

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.

Ein<em&gt; Ex-vivo-</em&gt; Modell um die Hormonwirkung in der menschlichen Brust Studieren

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.

Isolierung von Brustepithelzellen aus dreidimensionalen Mixed-Zelle Spheroid Co-Kultur

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