Ce travail a été soutenu par des subventions de recherche (NRF-2018-R1D1A1B07050175, HURF-2017-66) de la National Research Foundation (NRF) de Corée et du Hallym University Research Fund.

Tous les protocoles de recherche impliquant des souris de l’IC ont été approuvés par le Comité institutionnel de soins et d’utilisation des animaux (IACUC) de l’Université Yonsei au Wonju College of Medicine. Les expériences ont été réalisées conformément au Code d’éthique de l’Association médicale mondiale. Dans ce protocole, des souris enceintes d’ICR ont été maintenues dans un cycle clair/foncé de 12/12 h avec l’accès libre à la nourriture et à l’eau.
1. Dissection de Cochleae
<ol><li>Stérilisez la hotte de culture de tissu à flux laminaire en allumant la lumière ultraviolette pendant environ 30 min et vaporisez à toutes les surfaces de l’éthanol à 70% avant utilisation. Laissez les surfaces sécher.</li>
	<li>Placer les instruments de dissection dans de l’éthanol à 70% pendant 10 min et les sécher avant de les utiliser.</li>
	<li>Utilisez une lame d’opération pour décapiter des souris postnatales âgées de 3 à 4 jours(figure 2A).</li>
	<li>Placez le crâne sous un stéréomicroscope dans la hotte à flux laminaire et trempez le tissu dans de l’éthanol à 70%.</li>
	<li>Trempez rapidement le tissu dans une solution de dissection tissulaire (1x solution de sel équilibrée de Hank, HEPES 1 mM) pour éliminer l’éthanol.</li>
	<li>Couper l’axe du crâne à l’aide d’une lame chirurgicale(figure 2B,C).</li>
	<li>Exposer le crâne en tirant vers le bas de la peau vers le bas et en coupant le conduit auditif externe de l’oreille (Figure 2D).</li>
	<li>Couper de la partie antérieure à la partie postérieure du crâne à travers la ligne des yeux(figure 2E).</li>
	<li>Ouvrez le crâne et retirez le cerveau avant, le cervelet et le tronc cérébral à l’l’l’eau émoussée(figure 2F,G).</li>
	<li>À l’aide de micro pinces, séparer la cochlée de l’os temporal (Figure 2H).</li>
	<li>Transférer la cochlée dans une boîte de Petri contenant une solution de dissection tissulaire.</li>
	<li>Disséquer soigneusement toute la capsule otique cochléaire, ne laissant que les tissus mous cochléaires internes(figure 2I,J).</li>
	<li>Maintenez le modiolus de la cochlée avec une pince et le conduit cochléaire avec une autre paire de pinces, et séparez lentement les deux tissus (Figure 2K).</li>
	<li>Enlevez la stria vascularis et la membrane tectoriale en les épluchant doucement (Figure 2L,M).</li>
	<li>Placez une lame de couverture en plastique stérilisée dans une nouvelle solution de dissection tissulaire, puis placez l’organe de Corti sur une lamelle de couverture de 9 mm de diamètre, en vous assurant que la membrane basilaire est orientée vers le bas(Figure 2N-P).</li>
	<li>Immobilisez le tissu en appuyant sur la membrane de Reissner et le tissu modiolus restant sur la lamelle de couverture avec des pinces (Figure 2N-P).</li>
	<li>Transférer la lamelle avec le tissu incorporé au centre d’une capsule confocale de 35 mm de diamètre.</li>
	<li>Placer le cylindre de clonage de verre sur la parabole, avec l’explant cochléaire placé au centre de la parabole, et ajouter 100 μL de milieu de culture explantaire (DMEM/F12, 10 % de sérum fœtal bovin (FBS), supplément de N2 à 1 %, ampicilline (10 μg/mL))10 à l’intérieur du cylindre(figure 2Q).</li>
	<li>La plaque 5 × 103 cellules de CSM marqués par des protéines fluorescentes vertes (GFP) dérivées de la moelle osseuse de souris dans 2 mL de milieu de culture (45 % de DMEM + 45 % de DMEM/F12, 10 % de FBS, 1 % de supplément de N2, 10 μg/mL d’ampicilline) à l’extérieur du cylindre de verre(figure 2R).<ol>
<li>Lorsque les CSM sont confluents à 80-90%, faites-les passer en les détachant avec de l’acide tétraacétique trypsine-éthylènediamine.</li>
	</ol>
</li>
	<li>Transférer soigneusement le plat confocal dans un incubateur humidifié et incuber pendant la nuit à 37 °C dans une atmosphère à 5 % deCO2.</li>
	<li>Aspirez tout le milieu à l’intérieur et à l’extérieur du cylindre, puis retirez le cylindre en verre de la parabole confocale.</li>
	<li>Ajouter 2 mL de milieu de culture frais au plat confocal et incuber le plat de culture tissulaire dans un incubateur humidifié jusqu’à ce qu’il soit prêt pour l’analyse.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61947/61947fig2v2.jpg"> Figure 2. Dissection d’une cochlée de souris et coculture de l’organe de Corti et mscs. (A) Décapitation de la souris, (B) et (C) dissection sagittale médiane de la tête, (D) et (E) dissection coronale du cerveau, (F) et (G) ablation du cerveau et de l’os temporal, (H) la cochlée, (I) ablation de la paroi cochléaire osseuse, (J) isolement de la cochlée, (K) séparation du canal cochléaire du modiolus, (L) séparation de la stria vascularis (SV) et du ligament spiralé (SL) de l’organe de Corti, (M) ablation de la membrane tectoriale, (N-P) fixation de la cochlée sur un glissement de couverture en plastique, (Q) emplacement de la lame de couverture et du cylindre de verre dans la boîte confocale, (R) inoculation des CSM. Barre à écaille blanche (A-E) = 1 cm; barre d’échelle orange(F, G, P)et jaune(H,I)= 1 mm; barre d’échelle verte (J-O) = 0,5 mm. Veuillez cliquer ici pour voir une version plus grande de <a href="https://www.jove.com/files/ftp_upload/61947/61947fig2v2large.jpg" target="_blank">cette figure.</a>
2. Imagerie time-lapse
<ol><li>Pour les expériences présentées ici, utilisez un système de microscopie confocale avec un système d’incubateur à étage supérieur.</li>
	<li>Allumez le microscope confocal, la lumière fluorescente et l’ordinateur.</li>
	<li>Réglez les conditions de l’incubateur de plateau placé sur la scène du microscope confocal à 37 °C et 5 % d’atmosphère deCO2.</li>
	<li>Placez l’échantillon de plat sur le récipient de fixation de la parabole, couvrez avec le couvercle de fixation de la parabole et fermez la chambre avec le couvercle supérieur du chauffage.</li>
	<li>Ajustez le zoom et la mise au point pour localiser l’organe de Corti et mscs dans le champ de vision.</li>
	<li>Ouvrez le logiciel de traitement d’image. Sous l’option localiser, sélectionnez un objectif Plan-Apochromat 20x (ouverture numérique 0,8) et une surface de culture 0,5x.</li>
	<li>Sous Acquisition, cliquez sur smart setup (Configuration intelligente) et sélectionnez EGFP.</li>
	<li>Ouvrez l’onglet du canal sous Acquisitionet réglez la puissance laser à 0,2%, le trou d’épingle à 44 μm,le gain principal à 750 Vet le gain numérique à 1,0.</li>
	<li>Cliquez sur ESID sous configuration d’imagerie, et définissez le gain ESID sur 4 et le gain numérique sur 7.5.</li>
	<li>Cliquez sur Tuiles et jalonner pour produire 210 tuiles.</li>
	<li>Ouvrez la stratégie focus et sélectionnez le mode focus.</li>
	<li>Sous série chronologique, définissez la durée à 24 h et l’intervalle à 10 min.</li>
	<li>Sous Acquisition, définissez la taille de trame sur 512 x 512 pixels, la vitesse de balayage sur 8, la direction à bidirectionnelle, la moyenne à 4xet les bits par pixel à 16.</li>
	<li>Cliquez sur Démarrer l’expérience pour commencer l’expérience.</li>
</ol>3. Modification du fichier image
<ol><li>Sous traitement, cliquez sur couture, et définissez la superposition minimale à 5% et le décalage maximal à 10%.</li>
	<li>Cliquez sur exporter le film, définir non compressé, et réglez la vitesse sur 7.5.</li>
</ol>4. Immunomarquage
<ol><li>Aspirer soigneusement le milieu et laver l’échantillon deux fois avec une solution saline tamponnée au phosphate (PBS) pendant 5 min.</li>
	<li>Fixer l’échantillon avec 4% de forcine dans pbs pendant 15 min, et laver l’échantillon 3 fois avec PBS pendant 5 min.</li>
	<li>Perméabiliser l’échantillon dans 0,1% Triton X-100 dans pbs pendant 10 min, et laver 3 fois avec PBS pendant 5 min.</li>
	<li>Ajouter 250 μL de réactif phalloïdine-iFluor 647 (dilution de 1:1000 dans du PBS) et incuber l’échantillon pendant 1 h à température ambiante sur le shaker.</li>
	<li>Laver l’échantillon 3 fois avec du PBS pendant 5 min.</li>
	<li>Transférer la lame de couverture sur la lame de verre et ajouter 2 gouttes de solution de montage.</li>
	<li>Placez doucement une lame sur la glissière.</li>
	<li>Sceller la lamelle avec du vernis à ongles clair et conserver à 4 °C dans l’obscurité jusqu’à ce que les cellules soient observées.</li>
	<li>Imagez la lame à l’aide d’un microscope confocal avec un filtre approprié à excitation/émission (Ex/Em) = 650/665 nm pour la phalloïde et à Ex/ Em = 488/507 nm pour l’EGFP.</li>
</ol>

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+hearing+loss+prevention.">Global hearing loss prevention.</a> Otolaryngologic Clinics of North America. 51 (3), 575-592 (2018).</li><li>Chamberlain, G., Fox, J., Ashton, B., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25 (11), 2739-2749 (2007).</li><li>Fu, X., 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=Mesenchymal+stem+cell+migration+and+tissue+repair.">Mesenchymal stem cell migration and tissue repair.</a> Cells. 8 (8), (2019).</li><li>Uder, C., Br&#252;ckner, S., Winkler, S., Tautenhahn, H. 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La transplantation de CSM dans des sites endommagés pour favoriser la régénération des cellules endommagées a été intensivement étudiée, et l’effet thérapeutique est évident. On a rapporté que la transplantation et la différenciation subséquente des CSM rétablissent l’audition chez les rats présentant une perte auditive induite par l’acide 3 nitropropionique13. Bien que Lee et coll. aient appliqué des CSM aux êtres humains de façon transcanadienne, ils n’ont pas obtenu d...

La perte auditive peut survenir congénitalement ou peut être causée progressivement par plusieurs facteurs, y compris le vieillissement, les médicaments et le bruit. La perte auditive est souvent difficile à traiter car il est très difficile de restaurer une fonction altérée une fois que les cellules ciliées responsables de l’audition sont endommagées1. Selon l’Organisation mondiale de la santé, on estime que 461 millions de personnes dans le monde souffrent de perte auditive, ce qui représente 6,1 % de la population mondiale. Parmi les personnes ayant une perte auditive, 93 % sont des adultes et 7 % sont des enfants.
Un certain nombre d’approches ont été tentées pour traiter la perte auditive; notamment, une approche de régénération utilisant des CSM est apparue comme un traitement prometteur. Lorsque les tissus sont endommagés, les CSM sont naturellement libérés dans le système circulatoire et migrent vers le site de la blessure où ils sécrètent diverses molécules pour former un microenvironnement qui favorise la régénération2. Par conséquent, il est important de développer une méthode pour traiter les tissus endommagés par la migration des CSM implantés de l’extérieur vers les organes cibles et leur sécrétion ultérieure de molécules qui provoquent une régulation immunitaire puissante, l’angiogenèse et l’anti-apoptose pour améliorer la restauration de la fonction cellulaire endommagée3,4,5.
Le processus de homing dans lequel les CSM migrent vers les tissus endommagés peut être l’obstacle le plus important à surmonter. Les CSM ont un mécanisme de homing systémique avec des étapes séquentielles d’attache/roulement, d’activation, d’arrêt, de transmigration/diapédèse et de migration6,7,8. À l’heure actuelle, des efforts sont en cours pour trouver des moyens d’améliorer ces étapes. Diverses stratégies, y compris la modification génétique, l’ingénierie de surface cellulaire, l’amorçage in vitro et le guidage magnétique, ont été testées6,7. En outre, plusieurs tentatives ont été faites pour promouvoir la protection et la régénération des cellules ciliées auditives en homing MSCs au site de la cochlée endommagée. Cependant, le suivi des CSM in vivo prend du temps et exige beaucoup de main-d’œuvre et nécessite des compétences hautement spécialisées9.
Pour résoudre ce problème, une méthode a été développée pour observer le homing des CSM dans la cochlée par microscopie confocale time-lapse qui photographie la migration des cellules sur plusieurs heures(Figure 1). Il a été développé au début du 20 e siècle et est récemment devenu un outil puissant pour étudier la migration de cellules spécifiques.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> Figure 1: Résumé graphique ( A) Après que l’organe disséqué de Corti est collé sur une lame de couverture en plastique à l’aide d’une pince, la lame de couverture est placée sur une parabole microscopique confocale à fond de verre de 35 mm, et(B)le cylindre de verre est positionné. (C) Après avoir rempli l’intérieur du cylindre de verre avec un milieu, (D) les CSM marqués GFP avec du milieu sont ajoutés soigneusement à l’extérieur du cylindre. (E) Après une nuit d’incubation, (F) le cylindre de verre est retiré et les images sont prises avec un microscope confocal. Abréviations : GFP = protéine fluorescente verte; CSM = cellules souches mésenchymateuses. <a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">Veuillez cliquer ici pour voir une version plus grande de cette figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10X PBS Buffer</td>
			<td>GenDEPOT</td>
			<td>P2100-104</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Formalin</td>
			<td>T&#38;I</td>
			<td>BPP-9004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>sigma&#160;</td>
			<td>A5354-10ml</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>sigma&#160;</td>
			<td>A4503-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal dish</td>
			<td>SPL</td>
			<td>200350</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope&#160;</td>
			<td>ZEISS</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>coverslip</td>
			<td>SPL</td>
			<td>20009</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher scientific</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorsheild with DAPI</td>
			<td>sigma&#160;</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Dumont</td>
			<td>0508-L5-P0</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher scientific</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 647 Reagent</td>
			<td>abcam</td>
			<td>ab176759</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage Top Incubator</td>
			<td>TOKAI HIT</td>
			<td>WELSX</td>
			<td></td>
		</tr>
		<tr>
			<td>Strain C57BL/6 mouse messenchymal stem cells with GFP</td>
			<td>cyagen</td>
			<td>MUBMX-01101</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>sigma&#160;</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti

Pour étudier les effets des cellules souches mésenchymateuses (CSM) sur la régénération et le traitement cellulaires, cette méthode suit la migration msc et les changements morphologiques après co-culture avec l’épithélium cochléaire. L’organe de Corti a été immobilisé sur une lame de couverture en plastique en appuyant sur une partie du Reissner' membrane de s générée pendant la dissection. Les CSM confinés par un cylindre de verre ont migré vers l’épithélium cochléaire lorsque le cylindre a été retiré. Leur localisation prédominante a été observée dans le modiolus de l’organe de Corti, aligné dans une direction similaire à celle des fibres nerveuses. Cependant, quelques CSM ont été localisés dans le secteur de limbus et ont montré une forme horizontalement allongée. En outre, la migration dans le secteur de cellules ciliées a été augmentée, et la morphologie des CSM a changé à de diverses formes après traitement de kanamycin. En conclusion, les résultats de cette étude indiquent que la coculture des CSM avec l’épithélium cochléaire sera utile pour le développement de la thérapeutique par l’intermédiaire de la transplantation cellulaire et pour des études de régénération cellulaire qui peuvent examiner diverses conditions et facteurs.

La migration in vitro des CSM en mode tridimensionnel a été évaluée par un système Transwell ou par la méthode traditionnelle de cicatrisation des plaies pour observer la migration en mode bidimensionnel (2D)11. L’organe de Corti est une structure complexe composée de diverses cellules telles que les cellules de Boettcher, les cellules de Claudius, les cellules de Deiters, les cellules piliers, les cellules de Hensen, les cellules ciliées externes, les ce...

Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

Dans cette étude, nous présentons une méthode d’imagerie en temps réel utilisant la microscopie confocale pour observer les cellules se déplaçant vers le tissu endommagé par incubation ex vivo avec l’épithélium cochléaire contenant l’organe de Corti.

Imagerie in vitro de cellules vivantes en accéléré pour explorer la migration cellulaire vers l’organe de Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

in-vitro-time-lapse-live-cell-imaging-to-explore-cell-migration

Research

JoVE Journal

Medicine

3.7K vues.  Yonsei University Wonju College of Medicine. Dans cette étude, nous présentons une méthode d’imagerie en temps réel utilisant la microscopie confocale pour observer les cellules se déplaçant vers le tissu endommagé par incubation ex vivo avec l’épithélium cochléaire contenant l’organe de Corti.

Vidéo: In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Évaluation de la migration des cellules souches du cancer utilisant compartimenter dispositifs microfluidiques et imagerie des cellules vivantes

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Recherche

Médecine

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Modèles de MAME pour 4D imagerie des cellules vivantes de la tumeur: Interactions microenvironnement Progression impact malin

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

L&#39;utilisation de l&#39;IRMf pharmacologique-défi en recherche pré-clinique: Application à la System 5-HT

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Imagerie time-lapse de primaires prénéoplasiques cellules épithéliales mammaires issues de modèles de souris génétiquement modifiées de cancer du sein

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

L&#39;utilisation des puces à protéines de phase inversée (RPPA) Variation à explorer l&#39;expression des protéines dans les cancers à cellules rénales individuels

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

Confocale Accéléré Imaging comme une méthode efficace pour l&#39;évaluation cytocompatibilité de composites dentaires

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Cathétérisme cardiaque chez la souris pour mesurer le volume de pression Relation: Étudier les effets Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Modèle de souris de diabète de type 2: pancréatique isolement des îlots et des cellules vivantes 2-Photon Imaging d&#39;îlots intacts

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Through the Looking Glass: Time-lapse Microscopie et le suivi longitudinal des cellules individuelles pour l&#39;étude Therapeutics Anti-cancer

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Time-lapse Extended Imagerie intravitale du temps réel multicellulaire Dynamics dans le microenvironnement de la tumeur

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...