A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).&#160;
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.

1. incubation des oeufs <ol><li> Obtenir 8 jours anciens oeufs spécifiques exempts d&#39;agents pathogènes embryonnés. </li><li> Placez les oeufs dans la rotation plateau d&#39;oeufs, estampillés extrémités tournées vers le haut, et placez le plateau tournant à l&#39;intérieur d&#39;un incubateur d&#39;œufs. Incuber les oeufs pendant 48 heures à 36 ° C et 50% d&#39;humidité. </li></ol> 2. La chute du CAM et ouverture de l&#39;Eggs <ol><li> Ramassez des oeufs de l&#39;incubateur. Placez les oeufs dans casier à œufs, estampillés vers le haut, et porter à hotte à flux laminaire. </li><li> Éteignez les lumières chambre et de capot. </li><li> Maintenez la fin estampillée de œuf légèrement à l&#39;candler d&#39;oeuf pour exposer le système vasculaire de la CAM ainsi que le sac d&#39;air. NOTE: Le candler d&#39;œuf est une source de lumière utilisée pour visualiser l&#39;embryon et le sac d&#39;air associé et le système vasculaire. </li><li> Placez une marque de crayon entre deux principaux vaisseaux sanguins. </li><li> Allumez les lumières et utiliser une punaise stérile de faire un trou à la pointe de l&#39;oeuf au-dessus du sac d&#39;air (fin estampillée) et un trou au PENCIL marque. Ne poussez pas la tige de poussée tout au long; un trou d&#39;environ 3 mm de profondeur suffira habituellement. </li><li> Éteindre les lumières. Appuyez sur la poire de sécurité, en appuyant sur l&#39;extrémité ouverte fermement contre le trou au-dessus du sac d&#39;air. Appliquer aspiration pour aspirer l&#39;air dans le trou à la marque de crayon, provoquant la CAM pour tomber loin de la coque au niveau de la marque de crayon. </li><li> Vérifiez que le sac d&#39;air est passé de la fin estampillé de l&#39;œuf dans le trou de crayon marquée en utilisant la Candler. Si elle n&#39;a pas, utilisez la tige de poussée pour faire deux trous légèrement plus profonde et puis re-appliquer une aspiration à l&#39;aide de l&#39;ampoule de sécurité. </li><li> Allume la lumière. Tenir l&#39;œuf dans une main, activez l&#39;outil rotatif Dremel avec une roue de coupure 15/16 pouces joint et faire deux coupes transversales juste assez profonde pour couper à travers la coque, mais pas assez profonde pour couper tout le chemin jusqu&#39;à la dépression CAM. Faire des coupes autour de 2 cm de longueur et 1 cm de distance les uns des autres. Faire des coupes assez large pour que un anneau de silicone de 1 cm de diamètre peut passer à traversmais plus mince que la largeur du ruban adhésif standard. </li><li> Ensuite, faire une entaille longitudinale et entre les extrémités des deux coupes transversales en effleurant la roue de coupure à la coque. </li><li> Faites glisser des pinces stériles sous la pièce de coque et parallèle à la coque. Prenez la pièce de coque avec la pince et enlever toute la pièce proprement. </li><li> Placez du scotch sur la nouvelle fenêtre ouverte, en veillant à plier une extrémité sur elle-même pour faciliter l&#39;enlèvement. </li><li> Placez les oeufs dans l&#39;incubateur de retour dans le plateau d&#39;oeufs (pas dans les racks d&#39;œufs en rotation) jusqu&#39;à ce moment de l&#39;inoculation. </li></ol> 3. Ensemencement <ol><li> Gardez mélange de protéines de la membrane basale, comme matrigel, sur de la glace (pour éviter la polymérisation) et placer dans la hotte. </li><li> Utilisez 2 mM EDTA pour détacher les cellules (4 - 5 ml par flacon T75) et placez flacons dans le tissu incubateur de culture pendant 5 min. </li><li> Remettre les cellules individuelles dans les médias et à compter en utilisant un hémocytomètre et bleu trypan. Utilisez entre 500.000 to 2.000.000 cellules pour greffage sur la came, en fonction de la lignée cellulaire. Optimiser le nombre approprié dans des expériences préliminaires. </li><li> Mesurer le volume nécessaire pour le numéro de cellulaire appropriée, et centrifuger à 500 g pendant 5 min. </li><li> Rejeter le surnageant et remettre en suspension puis culot cellulaire dans 40 μ l de PBS ++ et 20 μ l de mélange de la membrane basale. NOTE: A ce stade, les cellules peut être traitée avec des médicaments ou d&#39;autres agents de test que nécessaire pour les expériences en remettant en suspension dans la cellule / sous-sol mélange membrane suspension. Alternativement, les agents peuvent être à la pipette sur la cellule / sous-sol mélange membrane suspension après la greffe. </li><li> Récupérer casier à œufs avec des oeufs de l&#39;incubateur et le placer dans la hotte à flux laminaire. </li><li> Décollez la bande de la fenêtre, de récupérer un anneau de silicone 1 mm du bouchon d&#39;un flacon cryogénique en utilisant des pinces stériles, et déposer l&#39;anneau à travers la fenêtre. </li><li> Pipet la solution de PBS l cellulaire de 60 μ ++ et membrane basale mixture directement dans le centre de l&#39;anneau de silicone en appui sur la came. </li><li> Re-bande de la fenêtre et déplacer casier à œufs retour à l&#39;incubateur. À ce stade, faites attention en déplaçant les oeufs que l&#39;anneau et la suspension cellulaire peut facilement devenir des personnes déplacées. </li><li> Autoriser les tumeurs de croître de 5 à 7 jours. </li></ol> 4. Récolte tumeurs <ol><li> Placer sous-pad dans le capot. Préparer 35 x 10 mm et plaques de conteneurs de paraformaldéhyde comme nécessaire pour l&#39;histologie. Mettre 1 ml de PBS ++ dans chaque plaque 35 mm x 10. </li><li> Placez sac pour matières contaminées dans le capot. Prenez des ciseaux de dissection stériles et insérer l&#39;extrémité pointue dans l&#39;œuf au trou près de la fin estampillé. </li><li> Couper autour de l&#39;œuf entier longitudinalement. NOTE: le CAM et la tumeur avec la bague doivent tenir à la moitié supérieure de la coque avec la fenêtre. </li><li> Avec des ciseaux de dissection et forceps, disséquer la tumeur loin de la CAM et le placer dans la tumeur mm plaque 35 x 10. Mesurer et peser tumeurs. Tumeurs fixes dans paraformaldehypuis de les intégrer paraffine pour une utilisation dans des expériences d&#39;histologie et immunohistochimie. </li></ol> 5. (Facultatif): cellules tumorales dissociation <ol><li> Utilisez des ciseaux de dissection pour hacher la tumeur dans la plaque 35 mm x 10. Verser la tumeur hachés dans un tube de 15 ml centrifugeuse et enlever autant PBS ++ que possible par pipetage. </li><li> Ajouter 2 ml de collagénase (1 mg / ml dans du PBS ++), tourner le tube à plusieurs reprises, et incuber le tube à 37 ° C pendant au moins 30 min (jusqu&#39;à plusieurs heures). </li><li> Ajouter 5 ml de milieu à chaque tube et remettre soigneusement par aspiration et en baisse de 20 - 40x. Pipetage approfondie est essentiel pour la dissociation complète des cellules. </li><li> Laisser sédiments de se déposer et transférer le surnageant dans un nouveau tube. Centrifuger à 500 g pendant 5 min. Retirer le surnageant, laissant derrière culot cellulaire dissociée. NOTE: A ce stade, les culots cellulaires peuvent être utilisés pour diverses expériences (cytométrie de flux, la lyse pour une utilisation dans un Western blot, l&#39;isolement de l&#39;ARN, etc). Le nombre total de cellules tumorales peuvent également être obtenus en utilisant un hémocytomètre et coloration au bleu de trypan. Notez que des cellules plus petites, de forme ovale sont des cellules de poulet de la CAM et ne doivent pas être considérés comme des cellules tumorales. Il devrait y avoir un nombre minimal de contamination des cellules non tumorales (CAM) si la tumeur est disséqué loin de la CAM avec soin. </li></ol>

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A.S.&#160;receives&#160;funding from the Advaxis Pharmaceutical company to support an investigator-initiated clinical trial.&#160;

Plusieurs étapes clés de ce protocole représentent la plus probable pour l&#39;amélioration de la survie des embryons ainsi que la fiabilité accrue de la croissance tumorale. La chute du CAM loin de la coquille en appliquant une aspiration dans le sac d&#39;air est moins invasive que les autres méthodes existantes (à l&#39;aide d&#39;une aiguille pour enlever l&#39;albumine de l&#39;œuf, dissection, etc.). En utilisant une punaise stérile pour créer les deux petits trous nécessaires à cette méthode est la p...

Carcinome hépatocellulaire (CHC) est la 3 ème cause de mortalité par cancer dans le monde 1. Actuellement, seulement 30% des patients HCC sont admissibles à des traitements chirurgicaux potentiellement curatives 2, et la chimiothérapie systémique est pas efficace 3. Par conséquent, il ya un besoin urgent non satisfait clinique de nouvelles thérapies de HCC, et le développement de systèmes de modèles appropriés pour tester l&#39;efficacité de nouveaux agents. Le dosage chorioallantoïque poussin membrane (CAM) offre une méthode à débit moyen rentable, rapide et reproductible de tester des médicaments potentiels anti-tumorales in vivo. Le dosage de la CAM a été largement utilisée pour étudier l&#39;angiogenèse 4. Il a également été développé avec succès dans un modèle de xénogreffe de tumeur de cancers, y compris un glioblastome 5, 6 cancer du pancréas, le mélanome 09/07, 11/10 et l&#39;ostéosarcome. Les deux 12 in ovo et ex ovo13 techniques ont été utilisées dans la littérature, avec des détails variant de protocole pour le protocole. Un défi majeur pour le modèle de xénogreffe CAM est l&#39;incidence relativement élevée de la mort de l&#39;embryon après la manipulation de l&#39;œuf, avec des taux de mortalité de l&#39;embryon de poussin publiés allant de 25 à 50 pour cent 11-14. Dans cet article, nous décrivons le développement d&#39;un in ovo modèle de xénogreffe de CHC qui produit de manière fiable la croissance des trois dimensions, tumeurs vascularisées qui ressemblent histologiquement HCC indifférencié. Nous avons adapté un premier protocole décrit par Ossowski et al., 14 et avons réalisé chiches embryonnaire taux de jusqu&#39;à 93% de survie à la greffe extrêmement élevé de la tumeur.

<table border="0" cellpadding="0" cellspacing="0" width="1094">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Name of Reagent/ Equipment</td>
			<td style="width:108px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Premium incubated eggs</td>
			<td style="width:108px;">Charles River</td>
			<td style="width:119px;">N/A</td>
			<td>http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg incubators</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">Hova-Bator 2362N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Rotating egg trays</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">1611 Automatic Egg Turner</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg candler</td>
			<td style="width:108px;">Lyon</td>
			<td style="width:119px;">Hi-Power 950-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Dremel 100 rotary tool with 15/16 cut-off wheel</td>
			<td style="width:108px;">Dremel</td>
			<td style="width:119px;">100-N/7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Sterile forceps, push pin, dissection scissors, Scotch tape</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Matrigel</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">356234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Cryogenic vials, external thread with silicone washer</td>
			<td style="width:108px;">Corning</td>
			<td style="width:119px;">430659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the in ovo chick chorioallantoic membrane (cam) assay as an efficient xenograft model of hepatocellular carcinoma

La membrane chorioallantoïque poussin (CAM) commence à se développer au jour 7 après la fécondation et mûrit par jour 12. Le CAM est naturellement immunodéficientes et très vascularisée, ce qui en fait un système idéal pour l&#39;implantation de la tumeur. En outre, la CAM contient des protéines de la matrice extracellulaire comme la fibronectine, la laminine, le collagène, l&#39;intégrine alpha (v) beta3, et la MMP-2, ce qui en fait un modèle attractif pour étudier l&#39;invasion tumorale et les métastases. Les scientifiques ont longtemps profité de la physiologie de la CAM en utilisant comme modèle de l&#39;angiogenèse. Plus récemment, le dosage de la CAM a été modifié pour fonctionner comme un système in vivo de modèle de xénogreffe de différents cancers qui comble le fossé entre le travail de base in vitro et des modèles de cancer chez les animaux plus complexes. Le dosage CAM permet l&#39;étude de la croissance tumorale, les thérapies anti-tumorales, et les voies moléculaires pro-tumorale dans un système biologiquement pertinent qui est à la fois en temps et en coût efficace. Ici, nous décrivons le développement de CAM Xenograft modèle de carcinome hépatocellulaire (HCC) avec le taux de survie embryonnaire de jusqu&#39;à 93% et fiable take tumeur conduisant à la croissance de tumeurs vascularisées, en trois dimensions.

Photos représentatives des étapes clés dans le protocole sont présentés ici. Figure 1A illustre l&#39;utilisation de l&#39;candler de visualiser le développement de l&#39;embryon, le sac d&#39;air, et le système vasculaire de la CAM. Figure 1B-1C montrent le processus de décrochage de la CAM en faisant les deux trous, puis en appliquant une pression négative en utilisant l&#39;ampoule de la sécurité, et la figure 1D montre un chuté succès C...

Watch this Scientific Journal Video about The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma at JoVE.com

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

La<em&gt; En Ovo</em&gt; Chick chorioallantoïque membrane (CAM) Assay comme un modèle de xénogreffe efficace de carcinome hépatocellulaire

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

the-ovo-chick-chorioallantoic-membrane-cam-assay-as-an-efficient

Research

JoVE Journal

Medicine

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

34.9K vues.  Icahn School of Medicine at Mount Sinai. The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

Vidéo: The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-photon imagerie de l&#39;invasion des cellules tumorales dans un modèle de souris orthotopique du carcinome épidermoïde oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Recherche

Médecine

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Un modèle de souris orthotopique de cancer de la thyroïde anaplasique

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Le<em&gt; En ovo</em&gt; CAM-test comme un Xenograftmodel pour le sarcome

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Génération de sous-cutanée et intra-hépatique droits de carcinome hépatocellulaire xénogreffes chez la souris immunodéficientes

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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 ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Un «Patient-Like&quot; Orthotopique Syngénique modèle de souris de carcinome hépatocellulaire Métastases

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

Conception des Guides chirurgicaux CAD/CAM pour Reconstruction maxillaire à l’aide d’une approche interne

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Mesure de la contractilité isométrique de l’artère mésentérique de souris à l’aide de fil myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

Prédire la réponse au traitement aux thérapies guidées par l’Image à l’aide de Machine Learning : un exemple pour le Trans-artériel traitement du carcinome hépatocellulaire

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...