Diese Arbeit wurde vom NIH Zuschüsse R01 CA107469, R01 CA125577 und U01CA154224 CG Kleer, der University of Michigan Cancer Center Support unterstützt.

1. Herstellung von Materialien und Kultur Medien <ol><li> Tauwetter Wachstumsfaktor reduziert (GFR) Matrigel bei 4 ° CO / N. </li><li> Warm up für komplette Medien erforderlich Zelllinie bei 37 ° C MCF10A-ATCC angegebenen Medien; Die HME-Ham F-12-Medium, ergänzt mit 5% fötalem Rinderserum, 1 ug / ml Hydrocortison, 5 ug / ml Insulin, 10 ng / ml epidermalen Wachstumsfaktor, und 100 ng / ml Cholera-Toxin; Die SUM149-Ham F-12 Medium mit 5% FBS, 2 &amp;mgr; g / ml Hydrocortison und 5 ug / ml Insulin; und MDA-MB-231-10% haben FBS-DMEM </li><li> Vorkühlung eine 8-Well-Kammer-Folie auf Eis. </li><li> Bereiten Gewebekultur Kapuze und Zubehör. </li></ol> 2. Dreidimensional Kultur in 8-Well Chamber Slide <ol><li> Mantel jedes Well der 8-Well-Objektträger mit 40 ul GFR Matrigel. Fügen Sie den Matrigel in der Mitte der Folie und dann verteilt mit einem P200 Pipettenspitze. Versuchen Sie, so gleichmäßig wie möglich zu verbreiten, die Vermeidung von Blasen und Überspreizung, die dazu führen könnenMeniskusbildung. </li><li> Die Objektträger bei 37 ° C unter 5% CO 2. Während die Matrigel verfestigt, trypsinize die Zellen, sammeln und Spin bei 150 xg in Gewebekulturzentrifuge für 4 min. </li><li> Zählen Sie die Zellen und mit Wasser auf 12.500 Zellen / ml in kompletten Medien der jeweiligen Zelllinie. In GFR Matrigel bis 2%, und fügen Sie 400 ul in jede Vertiefung einer Matrigel schwemmt Kammer Rutsche. </li><li> Inkubieren Sie die Folien in der CO 2-Inkubator. Geben frisch komplette Medien ändert sich jeden 4. Tag bis 15 Tage. </li><li> Behandlung mit pharmakologischen Inhibitoren: Bei Inhibitorstudien hinzuzufügen Kleinmolekül-Inhibitoren der vollständigen Medien zum Zeitpunkt der Beschichtung, gefolgt von frischem Medium wechselt mit den Inhibitoren ergänzt alle drei Tage während der Anzucht der Zellen auf Matrigel GFR. </li><li> Behandlung mit gereinigtem Protein: <ol><li> Platte die Zellen folgende Schritte 2.1-2.3. Ermöglichen, dass die Kulturen für 4 Tage, gefolgt von Serum wachsen starvatiauf 16 Stunden. </li><li> Am Tag 5, behandeln Sie die Zellen mit entsprechenden Konzentration von gereinigten Protein in 0,1% FBS Medien, um den Serumversorgten Zellen. Geben frisch Medienwechsel mit gereinigtem Protein nach 2 Tagen. </li><li> Am Tag 10 ersetzen den konditionierten Medien mit kompletter HME Medien. Lassen Sie die Kulturen für weitere 5 Tage wachsen. </li></ol></li><li> Immunfluoreszenzfärbung von auf Matrigel gewachsen acinar Strukturen: <ol><li> Herstellung von 2% Paraformaldehyd, 0,5% Triton X-100 und 100 mM Glycin in 1 × PBS, pH 7,4. Vorbereitung Immunpuffer (IF-Puffer), der aus 1x PBS, enthaltend 0,1% aufweist Rinderserumalbumin, 0,2% Triton X-100, 0,05% Tween-20. </li><li> Verwenden Sie ein P200 Pipettenspitze sorgfältig absaugen konditionierten Medien. </li><li> Befestigen Sie die acinar Strukturen mit 2% frisch zubereitet Para bei Raumtemperatur (RT) für 20 min. </li><li> Permeabilisierung der Zellen mit 0,5% Triton X-100 für 10 min bei 4 ° C. </li><li> Waschen Sie die Kulturs dreimal mit 100 mM Glycin, 10-15 Minuten für jede Waschung. </li><li> Blockieren der Zellen mit 10% Ziegenserum in IF-Puffer, genannt Primärblock, für 1 h bei RT. </li><li> Die Kulturen mit 20 &amp;mgr; g / ml Ziege-anti-Maus F (ab &#39;) 2-Fragment in der Grundblock-Puffer für 30 min inkubieren. Dieser Schritt ist wichtig, um die Maus-IgG immunreaktive Spezies in dem Matrigel blockieren. </li><li> Inkubieren mit primärem Antikörper in der zweiten Blockierlösung (Primärblock +20 ug / ml Ziege-anti-Maus F (ab &#39;) 2-Fragment) O / N bei 4 ° C. </li><li> Dreimal für jeweils unter leichtem Schütteln 10-15 min mit IF-Puffer waschen. </li><li> Inkubation mit fluoreszierenden konjugierten Sekundärantikörper in IF-Puffer für 45 min. </li><li> Wash 3x für jeweils 10 Minuten mit IF-Puffer unter leichtem Schütteln. </li><li> Montieren Sie die Folien mit Anti-Fade Reagenz, das DAPI, um die Kerne zu visualisieren. </li><li> Lassen Sie die Folien in O / N bei RT trocknen. </li><li> Bildaufnahme: Erfassung von konfokalen Bildern in der Kolonie midsection von Zellen auf Matrigel gewachsen. Für weitere Informationen zu erwerben eine Reihe von optischen Abschnitten über die gesamte Länge der Azinus-Struktur durch Z Stapeln. </li></ol></li></ol> 3. Dreidimensional Kultur in 2-Kammer und Objektträger für Immunoblotting <ol><li> Vorkühlung eine 2-Kammer und Schlitten auf Eis. Befolgen Sie die Schritte 2.1-2.4 Aufstockung der Reagenzien für die 2-Kammer auch Dia zB Mantel mit 160 ul Matrigel und fügen Sie 1,6 ml Zell / Matrigel mischen, um jede Vertiefung der 2-und Rutsche. </li><li> Ernten Sie die acinar Strukturen aus dem Matrigel Verwendung des kommerziell erhältlichen 3D-Kultur der Zellernte Kit folgenden Anweisungen des Herstellers. </li><li> Verdünne die Zellen zu waschen und Zellernte Puffer 1x und Vorkühlung auf 4 ° C Waschen und Verdrängen von Matrigel sollten auf Eis durchgeführt werden. </li><li> 3x mit dem Zellwaschpuffer waschen acinar Strukturen. </li><li> Sammeln Sie die Acini in 15 ml Zentrifugenröhrchen mit Zellernte buffer durch 30 min Inkubation auf einem Schüttler bei 4 ° C, gefolgt Mix Suspension auch mit einer 1 ml Pipette. Dadurch wird die Matrigel in kleine Stücke brechen und die meisten der Zellen in der Zellernte-Puffer. </li><li> Pelletieren Sie die Zellen bei 200 × g in einer Kultur zentrifugiert. Entfernen Sie die Schwimmschicht aus Hydrogel aus den pelletierten Zellen mit einem P200 Pipettenspitze. </li><li> Resuspendieren der Lysate in SDS-Probenpuffer mit dem Kit bei. RIPA Lysepuffer können ebenfalls verwendet werden. </li></ol> 4. Quantifizierung von malignen vs Gutartige Brust Phänotyp <ol><li> Wachsen Zellen in dreifacher Ausführung für jede Behandlung Satz folgende Schritte 2.1-2.4. </li><li> Nehmen Phasenkontrastbilder von acinar Strukturen aus jeder Vertiefung bei 5X oder 10X Auflösung mit einem Phasenkontrastmikroskop zu einer Folie Schieber und einen Computer angeschlossen ist. Nehmen die Bilder durch Bewegen des Schiebers von einem Rahmen zum anderen und somit über die gesamte Vertiefung der Kammerobjektträger. </li><li> Zählen Sie die Anzahl der acinar Strukturen,die Anzahl der einzelnen runden flachen Acini und der Anzahl der multiacinar Strukturen oder planlos gewachsenen Strukturen fehlen Organisation und Prozesse mit Verzweigung von ihr ausgeht. </li><li> Berechnen Sie den prozentualen Anteil der einzelnen runden flachen Strukturen acinar vs malignen Strukturen und Grundstück als Säulendiagramm. Berechnen Sie die Bedeutung von Student-t-Test. </li></ol>

<ol>
	<li>Arendt, L. M., Rudnick, J. A., Keller, P. J., Kuperwasser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroma+in+breast+development+and+disease.">Stroma in breast development and disease.</a> Semin. Cell Dev. Biol. 21, 11-18 (2009).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30, 256-268 (2003).</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>Li, Q., Chow, A. B., Mattingly, R. R. <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+overlay+culture+models+of+human+breast+cancer+reveal+a+critical+sensitivity+to+mitogen-activated+protein+kinase+kinase+inhibitors.">Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors.</a> J. Pharmacol. Exp. Ther. 332, 821-828 (2010).</li><li>Rosen, P. P., Rosen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rosen's breast pathology. , (2008).</li><li>Weaver, V. M., Fischer, A. H., Peterson, O. W., 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=The+importance+of+the+microenvironment+in+breast+cancer+progression:+recapitulation+of+mammary+tumorigenesis+using+a+unique+human+mammary+epithelial+cell+model+and+a+three-dimensional+culture+assay.">The importance of the microenvironment in breast cancer progression: recapitulation of mammary tumorigenesis using a unique human mammary epithelial cell model and a three-dimensional culture assay.</a> Biochem. Cell Biol. 74, 833-851 (1996).</li><li>Kenny, P. A., 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=The+morphologies+of+breast+cancer+cell+lines+in+three-dimensional+assays+correlate+with+their+profiles+of+gene+expression.">The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression.</a> Mol. Oncol. 1, 84-96 (2007).</li><li>Pal, A., Huang, W., Li, X., Toy, K. A., Nikolovska-Coleska, Z., Kleer, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCN6+modulates+BMP+signaling+via+the+Smad-independent+TAK1/p38+pathway,+acting+to+suppress+metastasis+of+breast+cancer.">CCN6 modulates BMP signaling via the Smad-independent TAK1/p38 pathway, acting to suppress metastasis of breast cancer.</a> Cancer Res. 15, 4818-4828 (2012).</li><li>Pal, A., Huang, W., Toy, K. A., Kleer, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCN6+knockdown+disrupts+acinar+organization+of+breast+cells+in+three-dimensional+cultures+through+up-regulation+of+type+III+TGF-&#946;+receptor.">CCN6 knockdown disrupts acinar organization of breast cells in three-dimensional cultures through up-regulation of type III TGF-&#946; receptor.</a> Neoplasia. 14, 1067-1074 (2012).</li><li>Wang, F., 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=Phenotypic+reversion+or+death+of+cancer+cells+by+altering+signaling+pathways+in+three-dimensional+contexts.">Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts.</a> J. Natl. Cancer Inst. 94, 1494-1503 (2002).</li><li>Weaver, V. M., 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=Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies.">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> J. Cell Biol. 137, 231-245 (1997).</li><li>Wang, R., 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=Three-dimensional+co-culture+models+to+study+prostate+cancer+growth,+progression,+and+metastasis+to+bone.">Three-dimensional co-culture models to study prostate cancer growth, progression, and metastasis to bone.</a> Semin. Cancer Biol. 15, 353-364 (2005).</li><li>Vaughan, M. B., Ramirez, R. D., Wright, W. E., Minna, J. D., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+model+of+differentiation+of+immortalized+human+bronchial+epithelial+cells.">A three-dimensional model of differentiation of immortalized human bronchial epithelial cells.</a> Differentiation. 74, 141-148 (2006).</li><li>Fata, J. E., Werb, Z., 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=Regulation+of+mammary+gland+branching+morphogenesis+by+the+extracellular+matrix+and+its+remodeling+enzymes.">Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes.</a> Breast Cancer Res. 6, 1-11 (2004).</li><li>Luca, A. 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=Impact+of+the+3D+microenvironment+on+phenotype,+gene+expression,+and+EGFR+inhibition+of+colorectal+cancer+cell+lines.">Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.</a> PLoS One. 8, 1-11 (2013).</li></ol>

Die Autoren erklären, keine finanziellen Interessen konkurrieren.

Wir haben hier die dreidimensionale Kultur von Brustepithelzellen auf einer rekonstituierten Basalmembran und die Nützlichkeit des Systems, zwischen bösartigen gegen gutartige Brust Phänotyp differen beschrieben. Dieses System kann auch zur Bildung verschiedener Zelltypen anderer Drüsengewebe verwendet werden, und es wurde berichtet, um erfolgreich zu Kultur Prostata-Epithelzellen, bronchiale Epithelzellen und Schilddrüsen-Epithelzellen verwendet haben, um nur einige zu nennen 12-14. Die Bedeutung der 3D...

Dreidimensionale Kultur (3D) von Brust-Epithelzellen auf rekonstituierte Basalmembran ist ein wichtiges Modellsystem, um die komplexe Phänotyp und zugehörige Signalisierung der normalen Brust und Brustkrebs 2,3 studieren. Die funktionelle Einheit der Brust ist der Endkanal lobulären Einheit (TDLU), die von einer kleinen Leitung, die Verzweigungen in Acini besteht. Die Acini sind hochorganisierten Strukturen, zwei Zellschichten, Epithel und Myoepithelzellen, die durch eine Basalmembran 5 umgeben sind, besteht. Die prägnantesten Merkmale des normalen Azini sind zelluläre Polarisation, Anbringung an der zugrunde liegenden Basalmembran und spezialisierten Zell-Zell-Kontakten. Dieses komplizierte Organisation ist in invasiven Karzinomen gestört. Die weit verbreiteten 2D Kulturen, in denen Zellen werden als Monoschichten gezüchtet, nicht für die Bildung der Acini zu ermöglichen. Somit wird die Monoschichtkultur fehlen in der Bereitstellung eines Systems, um die komplexen Beziehungen zwischen Epithelzellen zu erfassenin normalen Brust und ihre Deregulierung bei Brustkrebs. Funktionelle monotypic epithelialen Kulturen der Brustzellen wurden in mehreren Laboratorien 2,3 entwickelt. 3D-Kultur geht das Wachstum von Brust-Zellen auf Matrigel, die eine gelösten Extrakt aus Engelbreth-Holm-Swarm-Maus-Sarkom-Zellen abgeleitet ist und im Handel erhältlich. Andere 3D-Substrate wie Kollagen I werden ebenfalls verwendet. Die Brust-Zellen in 3D mit 3D-Embedded-Test, bei dem Zellen kultiviert in Matrigel 2 eingebettet sind kultiviert werden. Alternativ können Zellen durch 3D auf Assay kultiviert werden, die auch als 3D-Overlay-Assay, die kostengünstig ist, wie es erfordert weniger Volumen von Matrigel und erleichtert Zeitraffer-Überwachung der Koloniebildung durch Phasenkontrast-Bildgebung und ist ideal für in situ-Bildgebung 2 -3. Die 3D auf Test ist verwendet worden, um die Korrelation zwischen der acinar Phänotyp und Genexpressionsprofil 7 definieren. 3D-Kultur kann effektiver werdenEly verwendet, um normale und benignen Zellen von invasiven Karzinomen zu unterscheiden. Normal-und gutartigen Brustzellen bilden Wachstum verhaftet polarisierten Strukturen mit einem gut definierten Hohlraum in der Mitte auf der Erwägung, dass Matrigel invasiven Karzinomzellen überaus produktiv und planlos wachsen ohne Lichtung des Lumens. E6/E7 verewigt menschlichen Brust Epithelzellen und MCF10A Zelllinie, die eine spontan immortalisierte Zelllinie aus einem 36-jährigen Patienten mit Mastopathie isoliert, wurden erfolgreich eingesetzt, um die gutartige Brust Phänotyp in 3D 3,8 zurückzuerobern und wurden als serviert wird, ist ein Modellsystem, um die onkogene und Tumor-Suppressor-Funktion von mehreren Molekülen 8,9 studieren. Diese gutartigen Zellen können verändert werden, um Gen (e) von Interesse durch die Einführung von Lentiviren / Retrovirus manipulieren. Wenn das Gen (e) von Interesse ist ein Tumorsuppressor, werden shRNA-vermittelten Knockdown zu einer malignen Transformation wohin führen, wenn das Gen von Interesse ist ein Onkogen-Überexpression in benignen Zellensollte einen malignen Phänotyp zeigen. In beiden Fällen sind die in 3D gebildet acinar Strukturen können die maligne Transformation zu erfassen. Gutartige Einzelzellen in 3D vernickelt beginnen sich zu vermehren und bilden runde kugelförmige Strukturen. 5-8 Tage nach dieser sphärische Aggregat von Zellen wird in eine Umgebung polarisierte Schicht von Zellen, die in Kontakt mit dem Matrigel ist, und einer inneren Masse von weniger polarisierten Zellen, die Kontakt mit dem Matrigel fehlt unterscheiden. In den nächsten 10 bis 15 Tagen werden die inneren Zellen werden beginnen, durch Apoptose bildet eine klare Lumen sterben. Die malignen Zellen wachsen ihre Polarität planlos zu verlieren. Sehr bösartige Zellen bilden in der Regel Verzweigungsstrukturen. Diese Unterschiede im Phänotyp kann durch Zeitablauf Phasenkontrast-Bildgebung und durch in situ-Immunfärbung mit relevanten molekularen Marker erfasst werden. Die am häufigsten verwendeten Marker sind alpha 6-Integrin, eine basale Polaritätsmarkierung, in Verbindung mit dem Proliferationsmarker Phospho-Histon-3 und der apoptotic Marker Caspase-3 gespalten. Letzteres ist wichtig, um zu bestimmen, ob die Lumenbildung in der Mitte der Acini einem Merkmal der normalen und benignen Zellen. Andere Polarität und Zell-Zell-Kontakt Marker umfassen GM130, Laminin, ERM, E-Cadherin. Abwechselnd die Brustkrebszelllinien konstruiert werden, um das Gen von Interesse manipulieren. In diesem Fall wird eine Rückkehr zu mehr Wachstum verhaftet kann gutartig Phänotyp verwendet werden, wie ein ausgelesen, um von der Krebs-Phänotyp unterscheiden. 3D-Kultur kann auch verwendet werden, um sorgfältig die Signalwege in der malignen Transformation beteiligt 10-11 abzugrenzen. Mit Hilfe der oben genannten Lese-outs, pharmakologische Inhibitoren, blockierende Antikörper oder rekombinante Proteine ​​kann auf molekularer Signal die zu malignen Transformation ermitteln werden kann. Mit anhaltenden Bemühungen von verschiedenen Labors ist es möglich, die Zellen zu extrahieren, um aus 3D-RNA isolieren und bereiten auch Lysate für Immunoblotting und Immunnoprecipitation. Diese Strategien werden in einer schrittweisen und detailliert in dem Protokoll beschrieben.

<table><tbody><tr><td>Growth Factor Reduced Matrigel</td><td>BD Biosciences</td><td>354230</td><td>Avoid repeat freeze thaw&amp;nbsp;</td></tr><tr><td>Lab-Tek glass hamber slides 8-well</td><td>Thermo Fisher Scientific</td><td>177402</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Lab-Tek glass chamber slides 2-well&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>177380</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Goat anti mouse F(ab&amp;acute;)&lt;sub&gt;2 &lt;/sub&gt;fragment</td><td>Jackson Immunoresearch</td><td>115-006-006</td><td></td></tr><tr><td>Normal goat serum</td><td>Jackson Immunoresearch</td><td>005-000-121</td><td></td></tr><tr><td>Fluorescent conjugated Alexa antibodies</td><td>Molecular Probes</td><td></td><td>Please look at molecular probes website</td></tr><tr><td>Prolong gold antifade reagent with DAPI</td><td>Molecular Probes</td><td>P36935</td><td></td></tr><tr><td>3D Culture Cell Harvesting kit</td><td>Trevigen</td><td>3448-020-k</td><td></td></tr><tr><td>Anti-Apha-6-integrin</td><td>Fisher Scientific</td><td>MAB1378</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-GM130</td><td>BD Biosciences</td><td>610822</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-phospho-histone 3 (Ser10)FITC Conjugated</td><td>Fisher Scientific</td><td>16-222</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-Cleaved caspase 3 (Asp 175)</td><td>Cell Signaling</td><td>9661s</td><td>1:50 dilution for IF</td></tr><tr><td>Anti-E-cadherin</td><td>BD Biosciences</td><td>610181</td><td>1:200 dilution for IF</td></tr></tbody></table>

three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells

Invasive Mammakarzinome sind eine Gruppe von malignen epithelialen Tumoren gekennzeichnet durch die Invasion von benachbarten Geweben und Neigung zu metastasieren. Das Zusammenspiel von Signalen zwischen Krebszellen und ihrer Mikroumgebung übt einen starken Einfluss auf das Wachstum von Brustkrebs und biologischen Verhalten ein. Die meisten dieser Signale aus der extrazellulären Matrix verloren gehen oder ihre Relevanz under wird, wenn Zellen in einem zweidimensionalen Kultur (2D) als Monolayer gezüchtet. In den letzten Jahren hat dreidimensionale (3D) Kultur auf einer rekonstituierten Basalmembran als Methode der Wahl, um die Gewebearchitektur von benignen und malignen Brustzellen rekapitulieren entstanden. Zellen in 3D gewachsen behalten die wichtigen Signale aus der extrazellulären Matrix und einen physiologisch relevanten ex vivo-System 2,3. Zu beachten ist, gibt es immer mehr Hinweise darauf, dass Zellen verhalten sich anders, wenn im Vergleich zu 2D in 3D 4 gewachsen. 3D-Kulturkann effektiv als ein Mittel, um die malignen Phänotyp von der gutartigen Brust Phänotyp zu differenzieren und für die Untermauerung der zellulären und molekularen Signal verwendet werden 3 beteiligt. Eines der charakteristischen Merkmale von gutartigen epithelialen Zellen ist, dass sie polarisiert sind, so daß die apikale Zytoplasma zu dem Lumen und der basalen Zytoplasma ruht auf der Basalmembran. Diese apikal-basalen Polarität in der invasiven Mammakarzinome, die durch zelluläre Desorganisation und Bildung anastomosierenden und Verzweigung Tubuli, die planlos infiltriert das umgebende Stroma gekennzeichnet sind verloren. Diese histopathologischen Unterschiede zwischen gutartigen Drüse und invasives Karzinom in 3D 6,7 reproduziert werden. Mit den entsprechenden Lese-outs, wie die Quantifizierung der einzelnen Runde acinar Strukturen oder differentielle Expression von validierten molekulare Marker für Zellproliferation, Apoptose Polarität und in Kombination mit anderen molekularen und zellbiologischen Techniken, 3D-culturE kann ein wichtiges Instrument, um die zellulären Veränderungen während der malignen Transformation und für die Abgrenzung der zuständigen Melde besser zu verstehen ist.

3D Kultur effektiv zur malignen Phänotyp von menschlichen Brustepithelzellen von gutartigen Brust unterscheiden. Einzel benignen Zellen auf Matrigel vergoldet wachsen, wie organisiert kugelförmige Gebilde, die leicht als einzige runde, flache Acini in einer Ebene mit Phasenkontrast-Abbildung abgebildet werden kann. Die einzelnen malignen Zellen auf Matrigel vergoldet wachsen planlos, zeigen sehr hohen Brechungsindex und sind in einer Ebene schwer zu Bild. Wie in Fig. 1 gezeigt, ist die benigne HME und...

Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

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

Dreidimensionale Kultur von Säugetier-Epithelzellen auf einem rekonstituierten Basalmembran ist eine nützliche Methode, um die in vivo-Architektur der gutartigen Brust rekapitulieren und den malignen Phänotyp von gutartigen Brust Phänotyp zu differenzieren. Wichtiger ist, kann dieses System angewandt werden, um invasive Karzinome in anderen Geweben zu untersuchen.

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 ...

three-dimensional-cultures-tool-to-study-normal-acinar-architecture

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

19.8K Aufrufe.  University of Michigan Comprehensive Cancer Center.  Dreidimensionale Kultur von Säugetier-Epithelzellen auf einem rekonstituierten Basalmembran ist eine nützliche Methode, um die in vivo-Architektur der gutartigen Brust rekapitulieren und den malignen Phänotyp von gutartigen Brust Phänotyp zu differenzieren. Wichtiger ist, kann dieses System angewandt werden, um invasive Karzinome in anderen Geweben zu untersuchen. 

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

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Analyse der Kommunikation zwischen Monozyten und primären Brustkrebs-Zellen in einer extrazellulären Matrix (ECME) extrahieren-basiertes dreidimensionales System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Forschung

Krebsforschung

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Spheroid Assay zur TGF-β-induzierte Invasion Measure

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Medizin

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

Nachahmung und Manipulation von pankreatischen Acinar-duktale Metaplasie im 3-dimensionalen Zellkultur

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

In Vitro-Bewertung der onkogenen Transformation in menschlichen Mammaepithelzellen

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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 ...

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 ...

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 ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Herstellung und Analyse von<em&gt; In Vitro</em&gt; Three Dimensional Mammakarzinoms Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

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

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 ...