Le financement de ce projet est assuré par la Fondation pour la Recherche et Clayton Research Foundation Retina.

1. Préparation de tumorspheres rétinoblastome <ol><li> Obtenir un échantillon tumoral rétinoblastome dans un IRB-protocole approuvé. </li><li> Préparer une solution fraîche défini les médias de cellules souches (Neurobasal-A médias, 1X B-27 Supplément de moins de vitamine A, 1X non essentiels solution d&#39;acides aminés, 1X L-glutamine, 20 ng / ml EGF, 10 ng / ml de bFGF). </li><li> Mécaniquement désagréger le tissu tumoral avec un scalpel en utilisant une technique transversale dans un tissu stérile, la culture traitée à 6 puits assiette. Immédiatement ajouter 100 ul de définir les médias de cellules souches pour le tissu et écartez doucement les tissus hachés à l&#39;aide du scalpel. Ajouter 5 ml de définir les médias de cellules souches pour le bien. </li><li> Incuber à 37 ° C, 5% de CO 2 dans un incubateur humidifié et inspecter tous les jours. Certains tumorspheres devraient découler du tissu tumoral perturbé presque immédiatement, avec un nombre croissant au cours de 2-4 jours. </li><li> Hebdomadaire, tumorspheres centrifuger à 300xg pendant 5 minutes, remettre en suspension dans fraîchement préparés défini les médias de cellules souches, et une pipette de haut en bas 10 fois plus de perturber, de sphères et de permettre au centre nécrotique nouvelle, saine pour former des sphères. </li></ol> 2. Préparation des cellules tumorales pour l&#39;injection <ol><li> Si l&#39;injection de tumorspheres cultivés, doucement sphères de pipette dans un tube de 15 ml conique. Délicatement la pipette de haut en bas avec une pipette sérologique 10-15 fois à perturber les sphères. </li><li> Compter les cellules d&#39;être injecté à l&#39;aide d&#39;un hématimètre et aliquot au moins 50.000 cellules par injection dans un nouveau tube. Préparez-vous à des injections que possible en fonction du nombre de cellules disponibles. </li><li> Centrifuger à 300xg pendant 5 minutes. Aspirer délicatement le surnageant et remettre en suspension dans 5 ml de PBS. Répétez la centrifugation et de l&#39;aspiration, et remettre en suspension dans 10 pl de PBS par injection. </li></ol> 3. Préparation des animaux <ol><li> En utilisant une seringue à insuline, d&#39;administrer une injection intrapéritonéale de cocktail d&#39;anesthésie chez les rongeurs (kétamine 37,6 mg / mL, la xylazine 1,92 mg / mL, l&#39;acépromazine 0,38 mg / ml) à 1 l par gramme de poids corporel (GBW). Attendez 5 minutes pour que l&#39;animal devient un sédatif. Vérifier pouls et la place de l&#39;animal sur un tapis chauffant. Maintenir les animaux sur un tapis chauffant, à tout moment jusqu&#39;à la guérison. </li><li> Après la sédation, administrer une goutte de HCl proparacaïne à l&#39;oeil droit. Remplacer des animaux sur coussin chauffé et attendre 1 minute. </li><li> Administrer une goutte de chlorhydrate de phényléphrine à l&#39;oeil droit. Remplacer l&#39;animal sur le coussin chauffé et attendez 5 minutes. Si la dilatation de la pupille n&#39;a pas eu lieu, ré-administrer 1 goutte et d&#39;attendre encore 5 minutes. </li><li> Surveiller les animaux pour des signes de mouvement ou de tics, qui peut survenir à&gt; 30 minutes après la sédation. Dès les premiers signes de mouvement, d&#39;administrer une ul / GBW de kétamine HCl (dilué à 10 mg / ml dans PBS) et attendez 5 minutes. </li></ol> 4. L&#39;injection de cellules tumorales <ol><li> Placez l&#39;animal anesthésié sous le microscope sur le côté, avec l&#39;œil droit vers le haut et avec la tête posée sur de la gaze et centré afin d&#39;obtenir un réflexe rouge de l&#39;œil droit du fond d&#39;œil (pupille doit être dilatée à cette époque) lorsqu&#39;on les observe au microscope . </li><li> Caler le globe à droite en appuyant doucement avec deux doigts simultanément sur les paupières et maintenez cette position de façon constante pour le reste de la procédure. </li><li> En utilisant une solution stérile de calibre 30 aiguille reliée à une seringue luer-lock, percer dans le monde latéralement à travers la conjonctive et la sclère adjacente à la cornée du limbe dans le domaine de la pars plana (figure 1) et seulement à travers la choroïde dans la cavité vitréenne. Retirez l&#39;aiguille de l&#39;ouverture. </li><li> Stériliser l&#39;aiguille Hamilton avec une compresse imbibée d&#39;alcool. Dessinez la suspension cellulaire dans la seringue Hamilton et insérer l&#39;aiguille dans l&#39;ouverture jusqu&#39;à l&#39;aiguille est visualisée à travers le microscope derrière l&#39;objectif dans le vitré près de la rétine. Avec l&#39;aide d&#39;une deuxième personne tout en maintenant l&#39;aiguille de façon constante dans cette position, enfoncez lentement le piston. Retirer l&#39;aiguille. Si nécessaire, utilisez un coton-tige pour absorber tout liquide de l&#39;ouverture. </li><li> Relâchez doucement la pression des paupières de fermer les paupières de la souris. </li><li> Administrer une goutte d&#39;hypromellose à chaque oeil et le retour des animaux à coussin chauffé pour la récupération. </li></ol> 5. Suivi des souris injectées et la récolte des tumeurs <ol><li> Quotidien, examiner l&#39;œil injecté de leucocorie (blanc papillaire réflexe) et / ou distension périoculaire qui devient généralement apparente 2-4 semaines après l&#39;injection. </li><li> Une fois la croissance tumorale est détectée, euthanasier des animaux en fonction de directives institutionnelles. </li><li> Si l&#39;œil et la tumeur sont couverts par les paupières, utilisez le scalpel pour faire deux incisions orthogonale à la fente palpébrale et soulever les rabats de peau. </li><li> Sous la visualisation au microscope stéréoscopique, faire une incision circulaire au niveau du limbe, la suppression de la cornée et le cristallin, et soigneusement disséquer le gros de la masse tumorale d&#39;autres tissus en utilisant tweenisateurs. Placez la tumeur dans stériles RPMI-1640 médias. </li><li> Utilisez des pinces pour tirer lentement le globe ouverte de la douille. Afin de garantir un nerf optique, de sorte attachés invasion tumorale peuvent être visualisées, tirer le globe jusqu&#39;à ce que le nerf est exposée et utiliser des ciseaux pour couper le nerf le plus longtemps possible. Placer dans formol à 10% pour le traitement régulier pour un examen histologique. </li></ol> 6. Les résultats représentatifs: Tumorspheres rétinoblastome va commencer à apparaître à partir des tissus désagrégés presque immédiatement car ils sont libérés de la masse tumorale. Dans 2-4 jours, plus tumorspheres commence à se former et va augmenter en taille. Les sphères ont tendance à être régulières et présentent une stratégie bien définie, membrane secondaire entourant l&#39;ensemble (figure 2). L&#39;animal présente généralement leucocorie dans les 4 semaines après l&#39;injection (figure 3B), suivie par l&#39;élargissement de la planète et la distension des tissus environnants 5-8 semaines après l&#39;injection que la tumeur se développe (figure 3C). <img src="/files/ftp_upload/2644/2644fig1.jpg" alt="Figure 1"/> Figure 1. Transversales diagramme de l&#39;oeil de la souris en soulignant les caractéristiques mentionnées dans le protocole. <img src="/files/ftp_upload/2644/2644fig2.jpg" alt="Figure 2"/> Figure 2. Culture de cellules humaines in vitro rétinoblastome imagée à un 4x), et b) grossissement de l&#39;objectif 10x. Le rétinoblastome cellules tumorales primaires produisent tumorspheres avec une forme régulière sphéroïde et une membrane extérieure croustillante. Barres d&#39;échelle représentent un 500) um, et b) 200 um. <img src="/files/ftp_upload/2644/2644fig3.jpg" alt="Figure 3"/> Figure 3 Oeil de Rag2 -. / - Souris montrant une des caractéristiques) normale, b) indicatifs d&#39;une masse tumorale dans la cavité vitréenne leucocorie, et c) d&#39;une masse tumorale importante remplissant le globe avec une distension périoculaire associée, une hémorragie intra-oculaire.

<ol>
	<li>Ch&#232;vez-Barrios, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+and+Nonmetastatic+Models+of+Retinoblastoma.">Metastatic and Nonmetastatic Models of Retinoblastoma.</a> Am. J. Pathol. 157, 1405-1412 (2000).</li><li>Suber, M. L., Hurwitz, M. Y., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+consequences+of+intraocular+administration+of+modified+adenoviral+vectors.">Immune consequences of intraocular administration of modified adenoviral vectors.</a> Hum. Gene Ther. 12, 833-838 (2001).</li><li>Mallam, J. N., Hurwitz, M. Y., Mahoney, T., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Gene+Transfer+into+Retinal+Cells+Using+Adenoviral+Vectors:+Dependence+on+Receptor+Expression.">Efficient Gene Transfer into Retinal Cells Using Adenoviral Vectors: Dependence on Receptor Expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1680-1687 (2004).</li></ol>

Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements énoncés par le Baylor College of Medicine IACUC Comité.

La technique décrite ici facilite la propagation des tumeurs du rétinoblastome dans leur intraoculaire, le milieu intra-vitréenne. La technique d&#39;injection intra-oculaire a été utilisé dans le passé pour créer des lignées cellulaires de tumeurs du rétinoblastome dérivés 1 ainsi que pour fournir des vecteurs viraux pour la thérapie génique intraoculaire 2,3. Cette technique a maintenant été utilisé avec succès pour produire des tumeurs du rétinoblastome humain par injection directe de cellules de la tumeur primaire et l&#39;injection de tumorspheres ainsi que la propagation de série de tumeurs de xénogreffe. Preuves visibles de la formation de tumeurs (généralement leucocorie) est généralement d&#39;abord noté dans les 4 semaines, après quoi une hémorragie intra-oculaire et une distension du globe et / ou les tissus environnants de l&#39;orbite de développer dans les 5-8 semaines. Une minorité de souris injectées blesser l&#39;œil injecté, conduisant à la fermeture permanente de la paupière. Dans ces cas, la distension est le seul signe de la formation de tumeurs. Création de tumeurs murines xénogreffe n&#39;a pas été réussie avec l&#39;ensemble des tumeurs du rétinoblastome humain, si la propagation des tumeurs xénogreffe établie est très réussie. Cette observation suggère que certaines caractéristiques de la tumeur primaire, comme envahissant et le niveau de différenciation ou d&#39;autres facteurs inconnus, peuvent influencer la capacité de ces tumeurs à persister dans l&#39;environnement oculaires murin. La quantité de tissu qui peut être acquise à partir d&#39;une tumeur du rétinoblastome humain est assez petit, et il ya des limites importantes sur l&#39;aptitude à cultiver des cellules humaines in vitro rétinoblastome tels que la durée de vie limitée, la perte de l&#39;histologie des tumeurs solides et des changements dans le phénotype cellulaire. Ce protocole constitue un moyen relativement simple de propager la tumeur humaine et d&#39;établir un modèle murin de la maladie humaine. Cela permet à d&#39;autres in vitro et in vivo d&#39;expérimentation de la biologie du rétinoblastome et plus l&#39;entretien de l&#39;extérieur une tumeur du patient.

<table border="1"><tbody><tr><th> Nom </th><th> Société </th><th> Catalogue # </th><th> Commentaires </th></tr><tr><td> Chlorhydrate de phényléphrine 2,5% </td><td> Bausch &amp; Lomb </td><td> 053-11 </td><td> La solution ophtalmique </td></tr><tr><td> 30-ga Needle </td><td> BD </td><td> 305128 </td><td> Conique régulier </td></tr><tr><td> 10 ml Seringue Luer-Lock </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> Seringue à insuline 3/10-cc </td><td> BD </td><td> 328431 </td><td></td></tr><tr><td> Tampons alcoolisés </td><td> BD </td><td> 326895 </td><td></td></tr><tr><td> 6-Bien tissus Plate, de la Culture-traitées </td><td> BD </td><td> 353046 </td><td></td></tr><tr><td> Proparacaïne HCl 0,5% </td><td> Butler PAPA </td><td> 017239 </td><td> La solution ophtalmique </td></tr><tr><td> La kétamine HCl 100mg/mL </td><td> Fort Dodge </td><td> 4402A </td><td></td></tr><tr><td> 10 uL seringue Hamilton </td><td> Hamilton Company </td><td> 7648-01 </td><td></td></tr><tr><td> 32-ga Hamilton Needle </td><td> Hamilton Company </td><td> 7803-04 </td><td> Longueur sur mesure - 0.5 &quot; </td></tr><tr><td> Neurobasal-Médias </td><td> Invitrogen </td><td> 10888-022 </td><td></td></tr><tr><td> B-27 Supplément Minus vitamine A, 50X </td><td> Invitrogen </td><td> 12587-010 </td><td></td></tr><tr><td> RPMI-1640 avec les médias </td><td> Mediatech </td><td> 10 à 040-CV </td><td></td></tr><tr><td> Non-solution d&#39;acides aminés essentiels, 100X </td><td> Mediatech </td><td> 25 à 025-CI </td><td></td></tr><tr><td> L-Glutamine, 100X </td><td> Mediatech </td><td> 25 à 005-CI </td><td></td></tr><tr><td> N ° 11 Scalpel jetable </td><td> Miltex </td><td> 4-411 </td><td></td></tr><tr><td> Combo anesthésie rongeurs </td><td> n / a </td><td> n / a </td><td> Formulation de la pharmacie en interne (la kétamine 37,6 mg / mL, la xylazine 1,92 mg / mL, l&#39;acépromazine 0,38 mg / mL) </td></tr><tr><td> Facteur de croissance humain recombinant épidermique (EGF) </td><td> StemCell Technologies </td><td> 02633 </td><td> Reconstituer à 10 ug / ml de solution concentrée </td></tr><tr><td> Recombinant Human facteur de croissance basique des fibroblastes (bFGF) </td><td> StemCell Technologies </td><td> 02634 </td><td> Reconstituer à 10 ug / ml de solution concentrée </td></tr><tr><td> SGD-75 Microscope Operation </td><td> Topcon Medical Systems </td><td> SGD-75 </td><td> Ce modèle a été abandonné </td></tr><tr><td> Formol à 10% </td><td> VWR </td><td> 95042-908 </td><td></td></tr></tbody></table>

establishment and propagation of human retinoblastoma tumors in immune deficient mice

La culture de cellules tumorales dans le rétinoblastome défini les médias cellules souches suscite tumorspheres primaires qui peuvent être cultivées et entretenues pour seulement un temps limité. Ces tumorspheres cultivés peuvent présenter nettement différents phénotypes cellulaires par rapport aux tumeurs d&#39;origine. La démonstration que les cellules cultivées ont la capacité de former de nouvelles tumeurs est important de s&#39;assurer que le modèle de cellules cultivées de la biologie de la tumeur d&#39;origine. Nous présentons ici un protocole pour la propagation des tumeurs du rétinoblastome humain in vivo utilisant Rag2 - / - immunitaire des souris déficientes. Cultured tumorspheres rétinoblastome humain de passage faible ou cellules obtenues à partir fraîchement récoltées tumeurs du rétinoblastome humain injecté directement dans la cavité vitréenne d&#39;yeux murin former des tumeurs dans les 2-4 semaines. Ces tumeurs peuvent être récoltées et d&#39;autres passages soit dans les yeux murins in vivo ou cultivées comme tumorspheres in vitro. Propagation a été effectuée avec succès pendant au moins trois passages établissant ainsi une source continue de tissus humains pour le rétinoblastome de nouvelles expérimentations. Wesley S. Bond et Lalita Wadhwa sont co-premiers auteurs.

Watch this Scientific Journal Video about Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice at JoVE.com

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Une méthode est décrite pour propager les tumeurs humaines chez la souris rétinoblastome. Les cellules tumorales sont injectées directement dans les yeux du système immunitaire chez les souris déficientes. Tumeurs secondaires ont été établies avec succès en utilisant les deux cellules directement récoltées à partir de tumeurs humaines et tumorspheres cultivés.

Création et propagation des tumeurs de l&#39;homme en rétinoblastome immunitaire chez les souris déficientes

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

establishment-propagation-human-retinoblastoma-tumors-immune

Research

JoVE Journal

Medicine

11.7K vues.  Baylor College of Medicine. Une méthode est décrite pour propager les tumeurs humaines chez la souris rétinoblastome. Les cellules tumorales sont injectées directement dans les yeux du système immunitaire chez les souris déficientes. Tumeurs secondaires ont été établies avec succès en utilisant les deux cellules directement récoltées à partir de tumeurs humaines et tumorspheres cultivés.

Vidéo: Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

La coloscopie flexible chez la souris pour évaluer la sévérité de la colite et de tumeurs colorectales l&#39;aide d&#39;un système validé endoscopique de notation

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Recherche

Médecine

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Induction du envahissantes Transitional cancer de la vessie cellulaire dans immunitaire humain MUC1 souris transgéniques intactes: un modèle pour le développement d&#39;immunothérapie

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo L&#39;imagerie optique de tumeurs cérébrales et l&#39;arthrite Utilisation fluorescent SAPC-DOPS nanovésicules

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Fémorale aspiration de moelle osseuse chez des souris en direct

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Création anatomique précise et reproductible intracrânienne xénogreffes de tumeurs cérébrales humaines

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Mise en place d&#39;épithéliales humaines Enteroids et Colonoids de tout le tissu et la biopsie

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Mise en place d&#39;un modèle de xénogreffe humain de myélome multiple dans le poulet pour étudier la croissance tumorale, l&#39;invasion et l&#39;angiogenèse

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induction de l’athérosclérose accélérée chez les souris : le modèle « Wire-Injury »

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Caractérisation des cellules immunitaires dans les tissus adipeux humains à l’aide de cytométrie en flux

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.