Ce travail a été soutenu par une subvention pour la recherche scientifique C (subvention n ° 19K09834 à K.N.) du ministère de l’Éducation, de la Science et de la Culture du Japon.

Toutes les procédures ont été effectuées conformément au protocole approuvé par le Comité d’éthique pour le bien-être humain. Tous les patients ont donné leur consentement éclairé écrit pour l’utilisation de leurs échantillons à des fins de recherche, et la collecte et l’utilisation de tissus pour cette étude ont été approuvées par le Comité d’éthique de la recherche sur le génome humain de l’Université Teikyo.
1. Isolement et culture de cellules souches du cancer de l’ovaire (OCSC) provenant de patientes atteintes d’un cancer de l’ovaire et d’ascite dans une enceinte de biosécurité de niveau 2
<ol><li>Isoler les cellules souches cancéreuses de l’ascite du cancer de l’ovaire humain obtenu par paracentèse. Prélever au moins 100 à 250 mL d’ascite chez les patients pour prélever suffisamment de cellules souches cancéreuses. De plus, évaluer les profils d’expression des marqueurs de cellules souches cancéreuses (c.-à-d. EpCAM, Calrétinin, CD133, CD44, CD45, ALDH1 et Oct4) et des marqueurs du cancer de l’ovaire (pAX-8, WT-1) par cytométrie de flux.<ol><li>Centrifuger l’ascite du cancer de l’ovaire humain à 300 x g pendant 10 min à température ambiante dans les 24 heures suivant l’aspiration de l’ascite.</li>
		<li>Retirer le surnageant et ajouter 2 mL de milieu OCSC et 8 mL de solution saline tamponnée Histodenz/phosphate à 30 % (PBS, pH 7,4).</li>
		<li>Préparer le milieu OCSC : supplément de CSEh StemPro, DMEM⁄F-12 avec de la L-glutamine (milieu GlutaMAX), 25 % de BSA, 100 μM de 2-mercaptoéthanol, 8 ng/mL DE FGF BASIC, 10 μM d’insuline et 20 μM Y-27632.</li>
		<li>Superposer délicatement 2 mL de milieu OCSC à la solution cellulaire à l’étape 1.1.2 dans un tube de 15 mL et centrifuger à 450 x g pendant 20 min à température ambiante dans un rotor à godet oscillant sans freinage.</li>
		<li>Transférer délicatement la couche OCSC (non perturbée à l’interphase) dans un nouveau tube de 15 mL à l’aide d’une pipette de transfert.</li>
		<li>Remplir avec du PBS jusqu’à 15 mL. Centrifuger à 300 x g pendant 5 min à température ambiante et retirer le surnageant.</li>
		<li>Resuspendre la pastille cellulaire dans un milieu OCSC et semer sur une capsule de culture à très faible attache; les cultures doivent être maintenues à 37 °C dans 5 % de CO2.</li>
		<li>Changez le support tous les trois jours. Laissez soigneusement reposer le plat de culture pendant environ 1 minute, jetez une partie du surnageant et ajoutez le nouveau milieu.</li>
	</ol></li>
	<li>Adoption des CSC<ol><li>Recueillir les OCSC dans un tube de 15 mL et centrifuger à 200 x g pendant 5 min à température ambiante.</li>
		<li>Retirer le surnageant, remplir de PBS et centrifuger à 200 x g pendant 5 min à température ambiante.</li>
		<li>Retirer le surnageant, ajouter 1 mL de la solution de décollement cellulaire constituée d’enzymes protéolytiques et collagénolytiques (p. ex. AccuMax) et incuber à 37 °C pendant 10 min.</li>
		<li>Bien mélanger par pipetage et incuber à 37 °C pendant 5 min. Assurez-vous que les cellules sont dans une seule suspension.</li>
		<li>Bien mélanger par pipetage, remplir de PBS et centrifuger à 300 x g pendant 5 min à température ambiante.</li>
		<li>Retirer le surnageant et remettre en suspension la pastille cellulaire dans un milieu OCSC pour l’ensemencement ultérieur sur des boîtes de culture à très faible fixation et le maintien à 37 °C dans 5 % de CO2.</li>
	</ol></li>
</ol>2. Culture de cellules endothéliales HUEhT-1
<ol><li>Passage des cellules HUEhT-1<ol><li>Retirer le milieu de la boîte de culture HUEhT-1 et laver les cellules avec du PBS.</li>
		<li>Ajouter 1 mL de trypsine à 0,025% et incuber pendant 3 min à température ambiante.</li>
		<li>Ajouter 5 mL de milieu de croissance cellulaire endothéliale 2, recueillir les cellules dans un tube de 15 mL et centrifuger à 200 x g pendant 5 minutes à température ambiante.</li>
		<li>Retirer le surnageant, remettre en suspension la pastille cellulaire dans un milieu HUEhT-1 et ensemencer les cellules sur des boîtes de culture enrobées de collagène, puis les maintenir à 37 °C dans 5 % de CO2.</li>
		<li>Changez le support tous les trois jours.</li>
	</ol></li>
</ol>3. Préparation de la plaque de coculture NICO-1 pour le test de formation de tubes à l’aide de cellules HUEhT-1
<ol><li>Assembler NICO-1 et l’enrober avec l’hydrogel à base de matrice extracellulaire (Matrigel Matrix).<ol><li>Assemblez un côté du NICO-1 en suivant les instructions du fabricant et conservez-le sur la glace.</li>
		<li>Couvrir la surface de NICO-1 avec 300 μL de PBS froid, puis retirer le tampon.</li>
		<li>Ajouter 300 μL d’hydrogel refroidi à base de matrice extracellulaire et incuber à 37 °C pendant 60 min.</li>
		<li>Pour équilibrer, immerger le filtre avec de l’éthanol à 100%, puis laver un filtre ICCP de 13 mm (0,6 μm) avec du PBS pendant 1 min.</li>
		<li>Assemblez le NICO-1, y compris les deux parties principales du corps A (chambre droite) et B (chambre gauche) ainsi que le joint torique et le filtre équilibré.</li>
	</ol></li>
</ol>4. Ensemencement des cellules HUEhT-1 et des CSC sur le système NICO-1
<ol><li>Préparer les suspensions cellulaires HUEhT-1 en trypsinisant les monocouches cellulaires et en remettant les cellules en suspension dans un milieu de croissance cellulaire endothéliale avec du sérum de veau fœtal à 2%.</li>
	<li>Ajouter 1,2 mL de suspension cellulaire (1,5 x 105 cellules) à chaque puits recouvert d’hydrogel à base de matrice extracellulaire.</li>
	<li>Ajouter 1,5 mL de CSCO cultivés pendant cinq jours à l’autre puits.</li>
	<li>Incuber NICO-1 à 37 °C dans 5% CO2; La formation de tubes peut être observée au microscope et la formation de réseaux sur un hydrogel à base de matrice extracellulaire mesurée au moyen du nombre de branches.</li>
</ol>

<ol>
	<li>Bray, 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=Global+cancer+statistics+2018:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA, a Cancer Journal for Clinicians. 68, 394-424 (2018).</li><li>Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+development+and+metastasis.">Ovarian cancer development and metastasis.</a> American Journal of Pathology. 177 (3), 1053-1064 (2010).</li><li>Lytle, N. K., Barber, A. G., Reya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+fate+in+cancer+growth,+progression+and+therapy+resistance.">Stem cell fate in cancer growth, progression and therapy resistance.</a> Nature Reviews Cancer. 18 (11), 669-680 (2018).</li><li>Dontu, G., 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=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes and Development. 17 (10), 1253-1270 (2003).</li><li>Lonardo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal/Activin+signaling+drives+selfrenewal+and+tumorigenicity+of+pancreatic+cancer+stem+cells+and+provides+a+target+for+combined+drug+therapy.">Nodal/Activin signaling drives selfrenewal and tumorigenicity of pancreatic cancer stem cells and provides a target for combined drug therapy.</a> Cell Stem Cell. 9 (5), 433-446 (2011).</li><li>Ricci-Vitiani, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+expansion+of+human+colon-cancer-initiating+cells.">Identification and expansion of human colon-cancer-initiating cells.</a> Nature. 445 (7123), 111-115 (2007).</li><li>Ohata, H., 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=Induction+of+the+stem-like+cell+regulator+CD44+by+Rho+kinase+inhibition+contributes+to+the+maintenance+of+colon+cancer-initiating+cells.">Induction of the stem-like cell regulator CD44 by Rho kinase inhibition contributes to the maintenance of colon cancer-initiating cells.</a> Cancer Research. 72 (19), 5101-5110 (2012).</li><li>Ishiguro, T., 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=Establishment+and+characterization+of+an+in+vitro+model+of+ovarian+cancer+stem-like+cells+with+an+enhanced+proliferative+capacity.">Establishment and characterization of an in vitro model of ovarian cancer stem-like cells with an enhanced proliferative capacity.</a> Cancer Research. 76 (1), 150-160 (2016).</li><li>Singh, S. K., 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=Identification+of+a+cancer+stem+cell+in+human+brain+tumors.">Identification of a cancer stem cell in human brain tumors.</a> Cancer Research. 63 (18), 5821-5828 (2003).</li><li>Zong, X., Nephew, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+stem+cells:+role+in+metastasis+and+opportunity+for+therapeutic+targeting.">Ovarian cancer stem cells: role in metastasis and opportunity for therapeutic targeting.</a> Cancers (Basel). 11 (7), 934 (2019).</li><li>Liz&#225;rraga-Verdugo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+and+its+role+in+angiogenesis+and+vasculogenic+mimicry+in+gastrointestinal+cancers.">Cancer stem cells and its role in angiogenesis and vasculogenic mimicry in gastrointestinal cancers.</a> Frontiers in oncology. 10, 413 (2020).</li><li>Renaud, J., Martinoli, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+insert+co-culture+system+of+two+cellular+types+in+the+absence+of+cell-cell+contact.">Development of an insert co-culture system of two cellular types in the absence of cell-cell contact.</a> Journal of Visualized Experiments. (113), e54356 (2016).</li><li>Richardson, S. 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=Intervertebral+disc+cell-mediated+mesenchymal+stem+cell+differentiation.">Intervertebral disc cell-mediated mesenchymal stem cell differentiation.</a> Stem Cells. 24 (3), 707-716 (2006).</li><li>Plotnikov, E. Y., 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=Cell-to-cell+cross-talk+between+mesenchymal+stem+cells+and+cardiomyocytes+in+co-culture.">Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture.</a> Journal of Cellular and Molecular Medicine. 12 (5), 1622-1631 (2008).</li><li>Sheng, H., 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=A+critical+role+of+IFN-gamma+in+priming+MSC-mediated+suppression+of+T+cell+proliferation+through+up-regulation+of+B7-H1.">A critical role of IFN-gamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1.</a> Cell Research. 18 (8), 846-857 (2008).</li><li>Csaki, C., Matis, U., Mobasheri, A., Shakibaei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+canine+mesenchymal+stem+cells+with+primary+bone-derived+osteoblasts+promotes+osteogenic+differentiation.">Co-culture of canine mesenchymal stem cells with primary bone-derived osteoblasts promotes osteogenic differentiation.</a> Histochemistry and Cell Biology. 131 (2), 251-266 (2009).</li><li>Aguirre, A., Planell, J. A., Engel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+bone+marrow-derived+endothelial+progenitor+cell/mesenchymal+stem+cell+interaction+in+co-culture+and+its+implications+in+angiogenesis.">Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis.</a> Biochemical and Biophysical Research Communications. 400 (2), 284-291 (2010).</li><li>Proffen, B. L., Haslauer, C. M., Harris, C. E., Murray, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+from+the+retropatellar+fat+pad+and+peripheral+blood+stimulate+ACL+fibroblast+migration,+proliferation,+and+collagen+gene+expression.">Mesenchymal stem cells from the retropatellar fat pad and peripheral blood stimulate ACL fibroblast migration, proliferation, and collagen gene expression.</a> Connective Tissue Research. 54 (1), 14-21 (2013).</li><li>Goers, L., Freemont, P., Polizzi, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+systems+and+technologies:+taking+synthetic+biology+to+the+next+level.">Co-culture systems and technologies: taking synthetic biology to the next level.</a> Journal of the Royal Society &amp; Interface. 11 (96), 20140065 (2014).</li><li>De Palma, M., Biziato, D., Petrova, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumour+angiogenesis.">Microenvironmental regulation of tumour angiogenesis.</a> Nature Reviews Cancer. 17, 457-474 (2017).</li><li>Burger, 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=Incorporation+of+bevacizumab+in+the+primary+treatment+of+ovarian+cancer.">Incorporation of bevacizumab in the primary treatment of ovarian cancer.</a> New England Journal of Medicine. 365, 2473-2483 (2011).</li><li>Goel, H., Mercurio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+targets+the+tumour+cell.">VEGF targets the tumour cell.</a> Nature Reviews Cancer. 13, 871-882 (2013).</li><li>Yu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+bevacizumab+and+murine+VEGF-A:+a+reassessment.">Interaction between bevacizumab and murine VEGF-A: a reassessment.</a> Investigative Ophthalmology and Visual Science. 49 (2), 522-527 (2008).</li></ol>

Les auteurs n’ont rien à divulguer.

Le protocole présenté décrit comment imiter le microenvironnement tumoral des OCSC dans un contexte in vitro. La composante principale de la méthode constitue le modèle de coculture hautement reproductible obtenu à l’aide du système NICO-1, un système de co-culture indirect Transwell. Bon nombre des modèles de coculture actuellement disponibles examinent les effets du contact direct cellule-cellule sur les populations cellulaires en coculture 12,13,14,15,16,17,18.<sup class="...

Le cancer de l’ovaire est la huitième tumeur maligne la plus fréquente chez les femmes dans le monde, avec environ 300 000 nouveaux diagnostics et environ 180 000 décès par an1. Au diagnostic initial, le cancer de l’ovaire présente souvent des symptômes graves, environ 75% des patientes étant déjà au stade III-IV. En conséquence, le taux de survie à 5 ans est de <30% et le taux de mortalité est le plus élevé parmi les cancers gynécologiques2, l’efficacité du traitement du cancer de l’ovaire étant fortement dépendante de facteurs cliniques tels que la réussite de la chirurgie de réduction tumorale, la résistance à la chimiothérapie et la récidive après le traitement initial.
Les tissus cancéreux de l’ovaire sont organisés hiérarchiquement, tous les composants tumoraux n’étant pas également capables de générer des descendants. Les seules cellules capables de s’auto-renouveler et de produire une population hétérogène de cellules tumorales sont considérées comme représentant des cellules souches cancéreuses (CSC)3. L’auto-renouvellement du SCC et l’initiation de la tumeur s’accompagnent de la promotion de l’angiogenèse pour remodeler leur microenvironnement tumoral dans le but de maintenir un créneau de soutien. Cependant, les modèles précédents ne pouvaient pas être utilisés pour les analyses in vitro en raison de la reproductibilité limitée de la culture de CSC dérivés d’échantillons cliniques en raison de la perturbation des sphéroïdes après plusieurs passages. Plus récemment, des méthodes expérimentales de culture de CSC à partir de patients ont été développées pour plusieurs applications 4,5,6,7. En particulier, en exploitant la caractéristique des CSC de croître en formant des sphéroïdes dans des plaques de fixation ultra-basses avec un milieu sans sérum, les CSC cultivés sont amenés à exprimer un marqueur de surface des cellules souches qui n’est pas exprimé dans les cellules tumorales normales avec un potentiel de différenciation multilignée 8,9.
Des données récentes ont montré que la persistance des (O)CSC ovariens dormants visualisés comme une dissémination au péritoine est associée à leur régénération sous forme de tumeurs récurrentes10. La compréhension des caractéristiques moléculaires et biologiques des OCSC peut donc permettre un ciblage et une éradication efficaces de ces cellules, entraînant une rémission potentielle de la tumeur. En particulier, on sait peu de choses sur les caractéristiques mécanistes cellulaires et moléculaires des rôles des CSC dans l’angiogenèse11. Par conséquent, dans le présent protocole, nous avons utilisé des OCSC dérivés de patients dans un cadre in vitro pour étudier la propriété angiogénique des cellules endothéliales en utilisant le modèle de co-culture, qui peut imiter le microenvironnement tumoral des CSC et des cellules endothéliales au site métastatique en milieu clinique. En fin de compte, comme la néovascularisation constitue un processus critique nécessaire pour soutenir la croissance tumorale et les métastases, une meilleure compréhension de son mécanisme permettra le développement d’une nouvelle thérapie de ciblage pour les OCSC au site métastatique.
Ici, nous présentons un protocole pour visualiser l’étape de néovascularisation entourant les CSC de manière chronologique. L’avantage du protocole est de permettre des investigations entièrement reproductibles à l’aide du système de co-culture 3D, NICO-1, permettant ainsi d’observer les effets sur les patients de la capacité d’initiation tumorale dérivée de l’OCSC au cours de l’angiogenèse des cellules endothéliales.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.025% Trypsin&#160;</td>
			<td>Thermo</td>
			<td>R001100</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Pipet</td>
			<td>Thermo</td>
			<td>170356N</td>
			<td></td>
		</tr>
		<tr>
			<td>1250 &#181;L Pipet tip</td>
			<td>QSP</td>
			<td>T112XLRS-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Nunc</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L Pipet tip</td>
			<td>QSP</td>
			<td>T110RS-NEW</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo (Gibco)</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pipet</td>
			<td>Thermo</td>
			<td>170366N</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuMAX</td>
			<td>Innovative Cell Technologies</td>
			<td>AM105</td>
			<td></td>
		</tr>
		<tr>
			<td>BioCoatTM Collagen I 60mm Dish</td>
			<td>Corning</td>
			<td>356401</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>KUBOTA</td>
			<td>2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6 Well Clear Flat Bottom Ultra Low Attachment Multiple Well Plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium 2&#160;</td>
			<td>PromoCell</td>
			<td>C-22011&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>WAKO</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-Basic</td>
			<td>Thermo (Gibco)</td>
			<td>PHG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Histodenz</td>
			<td>SIGMA</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>HUEhT-1 cell</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB1458</td>
			<td></td>
		</tr>
		<tr>
			<td>ICCP Filter 0.6 &#181;m</td>
			<td>Ginrei Lab.</td>
			<td>2525-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, human</td>
			<td>SIGMA (Roche)</td>
			<td>11376497001</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>PerkinElmer</td>
			<td>ARVO MX-flad</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Yokogawa</td>
			<td>CQ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NICO-1</td>
			<td>Ginrei Lab.</td>
			<td>2501-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPlate-96</td>
			<td>PerkinElmer</td>
			<td>6005290</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 Pipet</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Pipet</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo (Gibco)</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro hESC SFM</td>
			<td>Thermo (Gibco)</td>
			<td>A1000701</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipet</td>
			<td>FALCON</td>
			<td>357575</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>WAKO</td>
			<td>253-00513</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating the angiogenetic properties of ovarian cancer stem-like cells using the three-dimensional co-culture system, nico-1

Les cellules souches cancéreuses (CSC) résident dans une niche de soutien, constituant un microenvironnement composé de cellules stromales adjacentes, de vaisseaux et de matrice extracellulaire. La capacité des CSC à participer au développement de l’endothélium constitue une caractéristique importante qui contribue directement à la compréhension générale des mécanismes de tumorigenèse et de métastases tumorales. Le but de ce travail est d’établir une méthodologie reproductible pour étudier la capacité d’initiation tumorale des cellules souches du cancer de l’ovaire (OCSC). Ici, nous avons examiné le mécanisme de néovascularisation entre les cellules endothéliales et les OCSC ainsi que les changements morphologiques des cellules endothéliales en utilisant le modèle de co-culture in vitro NICO-1. Ce protocole permet de visualiser l’étape de néovascularisation entourant les OCSC de manière chronologique. La technique peut fournir un aperçu des propriétés angiogénétiques des OCSC dans les métastases tumorales.

Nous avons recueilli des liquides d’ascite obtenus auprès de patientes atteintes d’un cancer de l’ovaire avancé lors d’une intervention chirurgicale ou d’une paracentèse dans le but d’effectuer une culture stable à long terme pour les sphéroïdes. Ici, nous présentons des cas d’une culture sphéroïde à long terme de CSC ovariens appelés CSC1 et CSC2. Les deux lignées cellulaires portent le même diagnostic et les mêmes profils histologiques. Les rôles mécanistes...

Watch this Scientific Journal Video about Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1 at JoVE.com

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

Les cellules souches du cancer de l’ovaire (OCSC) sont responsables de l’initiation du cancer, de la récidive, de la résistance thérapeutique et des métastases. La niche vasculaire OCSC est considérée comme favorisant l’auto-renouvellement des OCSC, conduisant à la chimiorésistance. Ce protocole fournit la base pour établir un modèle reproductible de niche vasculaire OCSC 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 ...

evaluating-angiogenetic-properties-ovarian-cancer-stem-like-cells

Research

JoVE Journal

Biology

2.5K vues.  Teikyo University School of Medicine. Les cellules souches du cancer de l’ovaire (OCSC) sont responsables de l’initiation du cancer, de la récidive, de la résistance thérapeutique et des métastases. La niche vasculaire OCSC est considérée comme favorisant l’auto-renouvellement des OCSC, conduisant à la chimiorésistance. Ce protocole fournit la base pour établir un modèle reproductible de niche vasculaire OCSC in vitro.

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

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Salut-C: une méthode d&#39;étude de l&#39;architecture tridimensionnelle des génomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Recherche

Biologie

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purification cytométrie en flux des cellules de souris méiotique

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

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.

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 ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

Un outil analytique qui quantifie les changements de morphologie cellulaire en trois dimensions des images de fluorescence

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Utilisation à haute résolution tomodensitométrie pour visualiser la structure tridimensionnelle et la fonction des vaisseaux sanguins des végétaux

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imagerie réorganisation spatiale d&#39;une voie de signalisation MAPK Utilisation du système d&#39;expression transitoire du tabac

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Développement d&#39;un système d&#39;insertion Co-culture de deux types cellulaires en l&#39;absence de Cell-Cell Contactez

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Reconstruction tridimensionnelle de l’Architecture vasculaire de l’ovaire de souris de clarté affranchis Passive

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear : Un tissu méthode de compensation pour l’imagerie en trois dimensions du tissu adipeux

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Imagerie tridimensionnelle à résolution unicellulaire des Organoïdes intacts

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...