1. L&#39;établissement en trois dimensions co-cultures <ol><li> Le jour avant d&#39;établir des co-cultures, retirez le Matrigel du congélateur et le dégel de la glace dans un 4 ° C réfrigérateur pendant la nuit. </li><li> Le jour de l&#39;expérience, pour préparer le milieu de co-culture, qui correspond à la moyenne pour les cellules épithéliales à être utilisés (moyenne HMEC dans cet exemple: DME/F12 milieu avec 10% de sérum bovin veau, 5 pg / ml d&#39;insuline , 1 ug / ml d&#39;hydrocortisone et 1 ng / ml EGF). Gardez le support sur la glace au moins 1 heure avant de les mélanger avec Matrigel. </li><li> Pour diluer le Matrigel décongelé, déterminer la quantité totale souhaitée de Matrigel: moyen (300 ul par puits pour plaques à 24 puits) et ajouter la moitié de ce volume de glace froide moyennes HMEC dans un tube stérile sur la glace. Puis ajouter le même demi-volume total de Matrigel décongelé au tube pour obtenir une dilution 1:1. Garder le tube sur la glace. </li><li> Placer 50 uL de Matrigel: mélange de milieu mi-bien dans les puits désignés d&#39;une plaque de 24 puits etincuber pendant 10 min dans un incubateur à 37 ° C avec de l&#39;air humidifié et 5% de CO 2. </li><li> Ajouter un supplément de 450 pi de Matrigel: mélange de milieu dans les puits et incuber pendant 60 minutes. </li><li> Au cours de l&#39;incubation, préparer une suspension 1:1 de fibroblastes (ici fibroblastes de réduction de la h-TERT-immortalisées mammaires exprimant la GFP) et les cellules épithéliales (ici h-TERT-humaines immortalisées ligne mammaire des cellules épithéliales exprimant RFP) à une concentration de 0,5 x 10 5 cellules chacune dans 0,5 ml de milieu HMEC. </li><li> Après l&#39;étape d&#39;incubation de 60 minutes, faire tomber doucement la suspension de cellules sur la surface du gel, solidifié, et retourner la culture dans l&#39;incubateur. </li><li> Ne dérangez pas les cultures pendant 2 jours. Après cela, support peut être changé à chaque 2-3 jours par aspiration très légèrement support à partir du côté du puits et lentement le remplacement par du milieu frais HMEC. </li><li> Surveiller la formation sphéroïde quotidienne en microscopie. Les cultures sont généralement matures après 10-14 jours. L&#39;image commeappropriée en utilisant la lumière ou la microscopie à fluorescence. La taille et la longueur de projection Sphéroïde peut être mesurée en microscopie optique. </li></ol> 2. Isolement des cellules de co-cultures Sphéroïde <ol><li> Très doucement, pipeter la culture de haut en bas 10x en utilisant un stérile de 2 ml pipette en verre. Sinon, couper la pointe d&#39;une pointe de 1000 microlitre pipette en plastique avec des ciseaux stérilisés par autoclavage ou immersion dans l&#39;éthanol à 70% afin de pipeter la culture. Transférer la culture avec soit la pipette de verre ou la pointe de pipette en plastique à une coupe stérile 15 tube de centrifugeuse ml et centrifugeuse pendant 5 min à 2000 tours par minute à température ambiante. Les sphéroïdes cellulaires seront sédiments au fond. </li><li> Utilisez un embout de pipette Pasteur stérile pour enlever délicatement le Matrigel perturbé par le haut du tube centrifugé. </li><li> Ajouter 1 ml HMEC moyen des sphéroïdes granulés, pipeter haut et en bas 2-3 fois avec une pipette stérile de 2 ml en verre, et de transférer la culture d&#39;un puits dans un de 24 puitsplaque de culture tissulaire. </li><li> Autoriser les sphéroïdes de joindre à l&#39;antenne pendant au moins 48 heures avant de changer le milieu. Au fil du temps, les cellules seront quitter les structures sphéroïdales et de grandir sous forme de monocouches. </li><li> Passage de la cellule cultures lorsque les cellules atteignent 50% de confluence. En premier la culture d&#39;un plat de 35 mm (environ 2-4 jours), puis à un plat de 100 mm (environ 3-5 jours). Immédiatement avant chaque étape de passage, utilisez une pipette Pasteur stérile pour supprimer manuellement le plus grand nombre résiduels &quot;sphéroïdes fantômes» que possible. </li><li> Analyser les cellules par cytométrie en flux pour la pureté de la population à l&#39;aide appropriées marqueurs de surface cellulaire et d&#39;autres populations cellulaires distinctes différents par tri cellulaire par fluorescence, si nécessaire. Les cellules isolées sont maintenant prêtes pour une analyse plus poussée comme vous le souhaitez. </li></ol> 3. Les résultats représentatifs En trois dimensions sphéroïde co-cultures, les fibroblastes avec d&#39;ingénierie Tiam1 silencing induit l&#39;invasion accrue dans matrix par les cellules épithéliales mammaires (figure 1, comparer D, E, F à A, B, C). Fibroblastes avec conçus sur-expression de Tiam1 induire l&#39;invasion matrice diminué par la lignée cellulaire de cancer du sein SUM159 (Figure 1, comparer J, K, L à G, H, I). Une fois que la co-culture sphéroïdes ont été mis en place, les populations de cellules peuvent être isolées à partir de sphéroïdes par replaquage dans un contexte à deux dimensions, comme le montre la figure 2. Selon les taux de croissance relatifs des cellules testées, des populations pures peuvent émerger à travers une série de passages (comme le montre la figure 3) ou peuvent être triés à l&#39;aide des marqueurs de cellules appropriées. Après isolement de la 3D co-culture, les cellules épithéliales de conserver les propriétés conférées par la co-culture avec des fibroblastes sur des périodes de temps prolongées, ce qui permet amplement l&#39;occasion pour l&#39;analyse. Dans cet exemple, l&#39;exposition à Tiam1 déficientes fibroblastes induit une migration accrue en co-cultivées HMECs qui a persisté pendant une semaineprès avoir l&#39;isolement de la 3D co-culture (Figure 4). <img alt="Figure 1" src="/files/ftp_upload/3760/3760fig1.jpg" /> Figure 1. Imagerie du sphéroïde créé des co-cultures en microscopie optique et de fluorescence. Cultures sphéroïdes sont établis avec lignées de cellules épithéliales mammaires et des fibroblastes et a permis de croître en culture pendant 10-14 jours. AF: épithéliales = Une HMEC-mCherry cellules. GL: les cellules épithéliales = Une SUM159-mCherry cellules. AC, IG: fibroblastes = contrôle RMF-GFP cellules. DF: fibroblastes = RMF-GFP avec Tiam1 silence. JL: fibroblastes = RMF-GFP avec Tiam1 sur-expression. Les flèches indiquent les projections épithéliales invasion dans la matrice. <img alt="Figure 2" src="/files/ftp_upload/3760/3760fig2.jpg" /> Figure 2. Imagerie de série de cultures en microscopie optique et de fluorescence après l&#39;isolement de la culture Matrigel. Jour 0 représente l&#39;imagerie immédiatement après l&#39;isolement, les jours 1, 2 et5 indiquent jours après l&#39;isolement. La couronne de cellules qui quittent les augmentations de sphéroïdes au fil du temps. Finalement, un «fantôme» sphéroïde avec très peu de cellules reste (flèche). Sphéroïdes il reste très peu après le retrait fantôme et le premier passage. <img alt="Figure 3" src="/files/ftp_upload/3760/3760fig3.jpg" /> Figure 3. Analyse par cytométrie en flux des cellules après isolement de Matrigel co-culture. Après sphéroïde co-cultures ont mûri, les cellules sont isolées de la 3D co-culture avec le pipetage et de série repiquage. Aliquotes sont analysées par cytométrie en flux (dans cet exemple en utilisant la fluorescence verte et rouge de fluorescence pour identifier GFP-exprimer les fibroblastes et les mCherry-exprimant les cellules épithéliales, respectivement) pour déterminer les populations relatives des cellules épithéliales et des fibroblastes. <img alt="Figure 4" src="/files/ftp_upload/3760/3760fig4.jpg" /> Figure 4. La migration Transwell de HMECs persiste après isolatià partir de Matrigel co-culture. Une fois que les populations pures ont été obtenues grâce à la culture ou le tri de cellules à fluorescence, les cellules peuvent être analysées comme vous le souhaitez. Voici la migration des HMECs co-cultivées avec un groupe témoin (C) ou Tiam1 déficientes (SHT) à travers des fibroblastes transwells perforées a été évaluée aux semaines 2, 4 et 8 après l&#39;isolement de co-culture.

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100, 57-70 (2000).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>Kim, J. B. <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+tissue+culture+models+in+cancer+biology.">Three-dimensional tissue culture models in cancer biology.</a> Semin. Cancer Biol. 15, 365-377 (2005).</li><li>Li, H., Fan, X., Houghton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment:+the+role+of+the+tumor+stroma+in+cancer.">Tumor microenvironment: the role of the tumor stroma in cancer.</a> J. Cell Biochem. 101, 805-815 (2007).</li><li>Tse, J. C., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+metastasis:+epithelial-to-mesenchymal+transition+and+contribution+of+tumor+microenvironment.">Mechanisms of metastasis: epithelial-to-mesenchymal transition and contribution of tumor microenvironment.</a> J. Cell Biochem. 101, 816-829 (2007).</li><li>Bhowmick, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling+in+fibroblasts+modulates+the+oncogenic+potential+of+adjacent+epithelia.">TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia.</a> Science. 303, 848-851 (2004).</li><li>Bhowmick, N. A., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-stroma+interactions.">Tumor-stroma interactions.</a> Curr. Opin. Genet. Dev. 15, 97-101 (2005).</li><li>Havlickova, B., Biro, T., Mescalchin, A., Arenberger, P., Paus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+optimization+of+an+organotypic+assay+system+that+imitates+human+hair+follicle-like+epithelial-mesenchymal+interactions.">Towards optimization of an organotypic assay system that imitates human hair follicle-like epithelial-mesenchymal interactions.</a> Br. J. Dermatol. 151, 753-765 (2004).</li><li>Kilani, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cytotoxicity+of+gemcitabine+in+bladder+cancer+cell+lines.">Selective cytotoxicity of gemcitabine in bladder cancer cell lines.</a> Anticancer Drugs. 13, 557-566 (2002).</li><li>Seidl, P., Huettinger, R., Knuechel, R., Kunz-Schughart, L. A. <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+fibroblast-tumor+cell+interaction+causes+downregulation+of+RACK1+mRNA+expression+in+breast+cancer+cells+in+vitro.">Three-dimensional fibroblast-tumor cell interaction causes downregulation of RACK1 mRNA expression in breast cancer cells in vitro.</a> Int. J. Cancer. 102, 129-136 (2002).</li><li>Silzle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+fibroblasts+recruit+blood+monocytes+into+tumor+tissue.">Tumor-associated fibroblasts recruit blood monocytes into tumor tissue.</a> Eur. J. Immunol. 33, 1311-1320 (2003).</li><li>Bartling, B., Demling, N., Silber, R. E., Simm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferative+stimulus+of+lung+fibroblasts+on+lung+cancer+cells+is+impaired+by+the+receptor+for+advanced+glycation+end-products.">Proliferative stimulus of lung fibroblasts on lung cancer cells is impaired by the receptor for advanced glycation end-products.</a> Am. J. Respir. Cell Mol. Biol. 34, 83-91 (2006).</li><li>Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+fibroblasts+to+regulate+the+formation+of+three-dimensional+vessel-like+structures+from+endothelial+cells+in+vitro.">Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro.</a> Am. J. Physiol. Cell Physiol. 290, C1385-C1398 (2006).</li><li>Liu, T., Lin, B., Qin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+promoted+tumor+spheroid+invasion+on+a+microfluidic+3D+co-culture+device.">Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device.</a> Lab Chip. 10, 1671-1677 (2010).</li><li>Zengel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+therapy+for+synergic+inhibition+of+tumour+cell+invasion+and+tumour-induced+angiogenesis.">Multimodal therapy for synergic inhibition of tumour cell invasion and tumour-induced angiogenesis.</a> BMC Cancer. 10, 92-92 (2010).</li><li>Kunz-Schughart, L. A., Heyder, P., Schroeder, J., Knuechel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heterologous+3-D+coculture+model+of+breast+tumor+cells+and+fibroblasts+to+study+tumor-associated+fibroblast+differentiation.">A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation.</a> Exp. Cell Res. 266, 74-86 (2001).</li><li>Djordjevic, B., Lange, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+interactions+in+spheroids+maintained+in+suspension.">Cell-cell interactions in spheroids maintained in suspension.</a> Acta Oncol. 45, 412-420 (2006).</li><li>Hirschhaeuser, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+an+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: an underestimated tool is catching up again.</a> J. Biotechnol. 148, 3-15 (2010).</li><li>Hoevel, T., Macek, R., Swisshelm, K., Kubbies, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reexpression+of+the+TJ+protein+CLDN1+induces+apoptosis+in+breast+tumor+spheroids.">Reexpression of the TJ protein CLDN1 induces apoptosis in breast tumor spheroids.</a> Int. J. Cancer. 108, 374-383 (2004).</li><li>Paduch, R., Kandefer-Szerszen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+D,+tamoxifen+and+beta-estradiol+modulate+breast+cancer+cell+growth+and+interleukin-6+and+metalloproteinase-2+production+in+three-dimensional+co-cultures+of+tumor+cell+spheroids+with+endothelium.">Vitamin D, tamoxifen and beta-estradiol modulate breast cancer cell growth and interleukin-6 and metalloproteinase-2 production in three-dimensional co-cultures of tumor cell spheroids with endothelium.</a> Cell. Biol. Toxicol. 21, 247-256 (2005).</li><li>Timmins, N. E., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method.">Generation of multicellular tumor spheroids by the hanging-drop method.</a> Methods Mol. Med. 140, 141-151 (2007).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Hanging+Drop+Cell+Culture+Protocol+for+Generation+of+3D+Spheroids.">A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids.</a> J. Vis. Exp. (51), e2720-e2720 (2011).</li><li>Xu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+fibroblast+Tiam1+in+tumor+cell+invasion+and+metastasis.">The role of fibroblast Tiam1 in tumor cell invasion and metastasis.</a> Oncogene. 29, 6533-6542 (2010).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

La méthode présentée ici représente une approche simple pour analyser les effets des fibroblastes du stroma sur les cellules épithéliales, y compris les cellules tumorales. Il permet de directement l&#39;interaction cellule-cellule dans un contexte tridimensionnel pris en charge par la matrice extracellulaire de la membrane basale, tout en permettant une extraction facile des cellules de la matrice pour une analyse plus approfondie. Cela peut être appliqué à l&#39;étude de associé à une tumeur stroma, comme ...

<table border="1"><tbody><tr><td> Nom de réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> BD Matrigel </td><td> BD Biosciences </td><td> 356237 </td><td> Phénol-rouge gratuitement </td></tr><tr><td> Hyclone moyen classique liquide: DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td> Hyclone bovine de sérum de veau </td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td> Insuline </td><td> EMD Chemicals </td><td> 407709 </td><td> Dissoudre dans 0.005N HCl; filtre de 0,22 uM </td></tr><tr><td> Hydrocortisone </td><td> Sigma-Aldrich </td><td> H4001 </td><td> Dissoudre dans 50% EtOH; filtre de 0,22 uM </td></tr><tr><td> EGF </td><td> Sigma-Aldrich </td><td> E9644 </td><td> Dissoudre dans de l&#39;acide acétique 10 mM; filtre de 0,22 um </td></tr></tbody></table>

isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture

Bien que des efforts énormes ont été consacrés à l&#39;identification des voies de signalisation et des molécules impliquées dans les comportements de cellules normales et malignes 1-2, une grande partie de ce travail a été fait à l&#39;aide classique en deux dimensions des modèles de culture de cellules, qui permettent de la manipulation des cellules facile. Il est devenu clair que les voies de signalisation intracellulaires sont touchés par les forces extracellulaires, y compris la dimensionnalité et la tension superficielle de cellules 3-4. De multiples approches ont été prises pour développer des modèles tridimensionnels qui représentent plus précisément l&#39;architecture des tissus biologiques 3. Bien que ces modèles intègrent multi-dimensionnalité et les contraintes architecturales, l&#39;étude des effets conséquents sur les cellules est moins facile que dans la culture de tissus à deux dimensions en raison des limites des modèles et de la difficulté à extraire des cellules pour une analyse ultérieure. Le rôle important du microenvironnement autour des tumeurs dans la tumorigenèse et le comportement de tumeur is de plus en plus reconnue 4. Stroma tumoral est composé de plusieurs types de cellules et molécules extracellulaires. Au cours du développement tumoral il ya des signaux bidirectionnels entre les cellules tumorales et les cellules stromales 5. Bien que certains facteurs participant à la tumeur du stroma co-évolution ont été identifiés, il ya encore un besoin de développer des techniques simples à identifier systématiquement et d&#39;étudier l&#39;éventail complet de ces 6 signaux. Les fibroblastes sont les cellules les plus abondantes dans normales ou associé à une tumeur des tissus stromales, et de contribuer au dépôt et à l&#39;entretien de la membrane basale et les facteurs de croissance paracrines 7. De nombreux groupes ont utilisé trois systèmes de culture dimensions pour étudier le rôle des fibroblastes sur diverses fonctions cellulaires, y compris la réponse tumorale aux traitements, le recrutement de cellules immunitaires, des molécules de signalisation, la prolifération, l&#39;apoptose, l&#39;angiogenèse et l&#39;invasion 8-15. Nous avons optimisé une méthode simple pour le culesser les effets de fibroblastes mammaires sur les cellules épithéliales mammaires en utilisant un modèle de matrice extracellulaire disponible dans le commerce pour créer des cultures tridimensionnelles de populations de cellules mixtes (co-cultures) 16-22. La co-culture avec des cellules suite former des sphéroïdes avec le regroupement des fibroblastes à l&#39;intérieur et les cellules épithéliales largement à l&#39;extérieur des sphéroïdes et formant multicellulaires saillies dans la matrice. Manipulation des fibroblastes qui mène à invasivité des cellules altérées épithéliale peut être facilement quantifié par des changements dans le nombre et la durée des projections épithéliales 23. En outre, nous avons mis au point une méthode pour isoler les cellules épithéliales sur trois dimensions de co-culture qui facilite l&#39;analyse des effets de l&#39;exposition sur le comportement des fibroblastes épithéliale. Nous avons constaté que les effets de la co-culture persister pendant des semaines après l&#39;isolement de cellules épithéliales, ce qui permet amplement de temps pour effectuer des essais multiples. Cette méthode est adaptable à des cellules devariant potentiel malin et ne nécessite aucun équipement spécialisé. Cette technique permet une évaluation rapide des modèles cellulaires in vitro dans des conditions multiples, et les résultats correspondants peuvent être comparés à des modèles in vivo de tissus animaux ainsi que des échantillons de tissus humains.

Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

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

Une méthode simple est décrite pour analyser les effets de fibroblastes de tissus sur les cellules épithéliales associées. La combinaison de cette méthode et la culture de tissus en trois dimensions peut faciliter l&#39;analyse des cellules après l&#39;isolement de la 3D. La technique est applicable à des cellules plus ou moins de potentiel malin, permettant l&#39;étude systématique des effets de associé à une tumeur du stroma sur les cellules tumorales.

Isolement des cellules épithéliales mammaires à partir de trois dimensions de cellules mixtes Sphéroïde 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 ...

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16.0K vues.  Tufts Medical Center. Une méthode simple est décrite pour analyser les effets de fibroblastes de tissus sur les cellules épithéliales associées. La combinaison de cette méthode et la culture de tissus en trois dimensions peut faciliter l'analyse des cellules après l'isolement de la 3D. La technique est applicable à des cellules plus ou moins de potentiel malin, permettant l'étude systématique des effets de associé à une tumeur du stroma sur les cellules tumorales.

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

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Souris cellules épithéliales mammaires forme Mammospheres cours de la différenciation lactogène

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

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Biologie

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidiques Co-culture de cellules épithéliales et des bactéries d&#39;enquête sur les Soluble signal médiée par des interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

Mammaires épithéliales procédure de greffe

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolement de la chromatine par purification d&#39;ARN (CHIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolement et culture des cellules souches épithéliales dentaire de l&#39;adulte de souris Incisive

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Évaluation des propriétés angiogénétiques des cellules souches du cancer de l’ovaire à l’aide du système de co-culture tridimensionnel, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

Coloration et imagerie haute résolution de modèles organoïdes et sphéroïdes tridimensionnels

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

Isolement des cellules épithéliales du follicule dentaire humain

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...