Il finanziamento per questo progetto è fornito dalla Fondazione Clayton per la Ricerca e la Fondazione di Ricerca Retina.

1. Preparazione del tumorspheres retinoblastoma <ol><li> Ottenere tumore campione retinoblastoma sotto un IRB-approvato il protocollo. </li><li> Preparare fresca di supporto definito di cellule staminali (Neurobasal-A media, 1X B-27 integratore meno vitamina A, 1X non essenziali della soluzione di amminoacidi, L-glutamina 1X, 20 ng / mL di EGF, 10 ng / ml bFGF). </li><li> Meccanicamente disaggregare il tessuto tumorale con un bisturi con una trasversale tecnica in una sterile, il tessuto della cultura trattati con 6-pozzetti. Immediatamente aggiungere 100 ml di mezzi di comunicazione definite cellule staminali del tessuto e delicatamente diffondere il tessuto tritata con il bisturi. Aggiungere 5 ml di mezzi di comunicazione di cellule staminali definite al pozzo. </li><li> Incubare a 37 ° C, 5% di CO 2 in un incubatore umidificato e controllare tutti i giorni. Alcuni tumorspheres dovrebbe derivare dal tessuto tumorale interrotto quasi subito, con un numero sempre crescente nel corso di 2-4 giorni. </li><li> Settimanale, tumorspheres centrifugare a 300xg per 5 minuti, risospendere nei media staminali preparati al momento definito di cellule, e seminare su e giù per 10 volte per interrompere più grandi, sfere centrale necrotica e consentire nuove sfere sano di forma. </li></ol> 2. La preparazione delle cellule tumorali per iniezione <ol><li> Se l&#39;iniezione tumorspheres colta, dolcemente sfere in una pipetta da 15 ml tubo conico. Pipettare delicatamente su e giù con una pipetta sierologica 10-15 volte per distruggere le sfere. </li><li> Contare le cellule da iniettare utilizzando un emocitometro, e aliquota almeno 50.000 cellule per iniezione in un nuovo tubo. Prepararsi per le iniezioni maggior numero possibile in base al numero di cellule disponibili. </li><li> Centrifugare a 300xg per 5 minuti. Con attenzione aspirare il surnatante e risospendere in 5 ml di PBS. Ripetere centrifugazione e aspirazione e risospendere in 10 L PBS per iniezione. </li></ol> 3. Preparazione degli animali <ol><li> Utilizzando una siringa per insulina, somministrare un&#39;iniezione intraperitoneale di cocktail roditori anestesia (ketamina 37,6 mg / ml, xilazina 1,92 mg / ml, acepromazina 0,38 mg / ml) a 1 ml per grammo di peso corporeo (GBW). Attendere 5 minuti per l&#39;animale a diventare sedato. Controllare il battito cardiaco e posizionare l&#39;animale su un blocco riscaldato. Mantenere gli animali su un blocco riscaldato in ogni momento fino al recupero. </li><li> Dopo la sedazione, somministrare 1 goccia di HCl proparacaina per l&#39;occhio destro. Sostituire animali sul pad riscaldato e attendere 1 minuto. </li><li> Somministrare 1 goccia di fenilefrina HCl per l&#39;occhio destro. Sostituire l&#39;animale sul pad riscaldato e attendere 5 minuti. Se la dilatazione della pupilla non si è verificato, ri-somministrare 1 goccia e attendere altri 5 minuti. </li><li> Monitorare gli animali per i segni di movimento o spasmi, che può verificarsi in&gt; 30 minuti dopo la sedazione. Al primi segni di movimento, somministrare 1 ml / GBW di ketamina HCl (diluito a 10 mg / ml in PBS) e attendere 5 minuti. </li></ol> 4. Iniezione di cellule tumorali <ol><li> Posizionare l&#39;animale anestetizzato sotto il microscopio su un lato, con l&#39;occhio destro rivolto verso l&#39;alto e con la testa appoggiata su garza e centrato per ottenere un riflesso rosso dell&#39;occhio destro fondo (pupilla dilatata dovrebbe essere in questo momento) quando osservata attraverso il microscopio . </li><li> Puntellare il globo destra spingendo delicatamente verso il basso contemporaneamente con due dita sulle palpebre e mantenere questa posizione costante per il resto della procedura. </li><li> Usando una sterile da 30 gauge collegato a una siringa Luer-Lock, perforare il globo lateralmente attraverso la congiuntiva e la sclera adiacente al limbus corneale nella zona della pars plana (figura 1) e solo attraverso la coroide in camera vitreale. Rimuovere l&#39;ago dalla apertura. </li><li> Sterilizzare l&#39;ago Hamilton con un batuffolo di cotone imbevuto di alcool. Disegnare la sospensione cellulare nella siringa Hamilton e inserire l&#39;ago nel foro fino a quando l&#39;ago viene visualizzato attraverso il microscopio dietro l&#39;obiettivo nel vitreo vicino alla retina. Con l&#39;aiuto di una seconda persona mentre si tiene l&#39;ago stabilmente in questa posizione, spingere il pistone lentamente. Rimuovere l&#39;ago. Se necessario, utilizzare un batuffolo di cotone per assorbire qualsiasi liquido dall&#39;apertura. </li><li> Delicatamente rilasciare la pressione delle palpebre per chiudere le palpebre del mouse. </li><li> Somministrare 1 goccia di ipromellosa per ciascun occhio e tornare animale pad riscaldato per il recupero. </li></ol> 5. Monitoraggio di topi iniettati e la raccolta dei tumori <ol><li> Ogni giorno, esaminare l&#39;occhio iniettato per leucocoria (bianco riflesso papillare) e / o perioculare distensione che di solito si manifesta 2-4 settimane dopo l&#39;iniezione. </li><li> Una volta che la crescita del tumore viene rilevato, l&#39;eutanasia degli animali secondo le linee guida istituzionali. </li><li> Se l&#39;occhio e tumore sono coperti dalla palpebre, utilizzare il bisturi per fare due incisioni ortogonali alla fessura palpebrale e sollevare i lembi della pelle. </li><li> Sotto la visualizzazione utilizzando il microscopio stereoscopico, fare una incisione circonferenziale al limbus, eliminando la cornea e la lente, e con attenzione sezionare la maggior parte della massa tumorale da altri tessuti utilizzando tweezers. Posizionare il tumore in sterili RPMI-1640 media. </li><li> Utilizzare le pinzette per estrarre lentamente il globo aperto da presa. Per assicurare un nervo ottico collegato in modo invasione tumorale possono essere visualizzati, tirare il globo fino a quando il nervo è esposto e usare le forbici per tagliare il nervo più a lungo possibile. Posto in formalina al 10% per l&#39;elaborazione normale di esame istologico. </li></ol> 6. Rappresentante dei risultati: Tumorspheres retinoblastoma inizierà ad apparire dal tessuto disaggregati quasi immediatamente come sono liberate dalla massa tumorale. Entro 2-4 giorni, più tumorspheres inizierà a formare e aumento delle dimensioni. Le sfere tendono ad essere regolari e mostrano una ben definita, la membrana che circonda l&#39;aggregato secondaria (Figura 2). L&#39;animale si presenta tipicamente con leucocoria entro 4 settimane post-iniezione (figura 3b), seguita dall&#39;ampliamento del globo e la distensione dei tessuti circostanti 5-8 settimane dopo l&#39;iniezione come il tumore cresce (Figura 3c). <img src="/files/ftp_upload/2644/2644fig1.jpg" alt="Figura 1"/> Figura 1. Sezione trasversale dell&#39;occhio del mouse mettendo in evidenza le caratteristiche si fa riferimento nel protocollo. <img src="/files/ftp_upload/2644/2644fig2.jpg" alt="Figura 2"/> Figura 2. Retinoblastoma cultura di cellule umane in vitro ripreso a) 4x, e b) ingrandimento obiettivo 10x. Cellule del tumore primario retinoblastoma produrre tumorspheres con una forma regolare sferoidale e una membrana esterna croccante. Barre rappresentano una scala) 500 micron, e b) 200 micron. <img src="/files/ftp_upload/2644/2644fig3.jpg" alt="Figura 3"/> Figura 3 Eye of RAG2 -. / - Topo mostrando a) funzioni di normale, b) indicativi di una massa tumorale nella cavità vitrea leucocoria, e c) una massa tumorale grande riempiendo il mondo con distensione perioculare associati, emorragia intraoculare.

<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>

Gli esperimenti sugli animali sono stati eseguiti in conformità con le linee guida e dei regolamenti previsti dal Baylor College of Medicine IACUC Comitato.

La tecnica qui descritta favorisce i tumori nel loro propagazione retinoblastoma intraoculare, ambiente intravitreale. La tecnica di iniezione intraoculare è stata utilizzata in passato per creare i tumori da retinoblastoma derivate linee cellulari 1, nonché per fornire vettori virali per la terapia genica intraoculare 2,3. Questa tecnica è stata utilizzata con successo per la produzione di tumori retinoblastoma umano mediante iniezione diretta di cellule dal tumore primario e l&#39;iniezione di tumorspheres, nonché la riproduzione seriale di tumori trapiantati. La prova visibile della formazione del tumore (di solito leucocoria) di solito è notata la prima volta entro 4 settimane, dopo di che emorragia intraoculare e distensione del / mondo e tessuti circostanti o l&#39;orbita sviluppano entro 5-8 settimane. Una minoranza di topi iniettati ferire l&#39;occhio iniettato, con conseguente chiusura permanente delle palpebre. In questi casi, la distensione è l&#39;unico segno della formazione di tumori. Istituzione di tumori murini trapiantati non ha avuto successo con tutti i tumori retinoblastoma umano, anche se la propagazione dei tumori trapiantati stabilito è di grande successo. Questa osservazione suggerisce che alcune caratteristiche del tumore primario, come l&#39;invasività e il livello di differenziazione o di qualche altro fattore sconosciuto, può influenzare la capacità di questi tumori a persistere nell&#39;ambiente oculare murino. La quantità di tessuto che può essere acquisito da un tumore retinoblastoma umano è abbastanza piccola, e ci sono significative limitazioni sulla capacità di cellule di retinoblastoma cultura umana in vitro ad esempio come span durata limitata, perdita di istologia tumore solido e cambiamenti nel fenotipo cellulare. Questo protocollo fornisce un modo relativamente semplice per propagare il tumore umano e stabilire un modello murino della malattia umana. Questo permette inoltre in vitro e in vivo la sperimentazione della biologia di retinoblastoma e più manutenzione del tumore al di fuori del paziente.

<table border="1"><tbody><tr><th> Nome </th><th> Azienda </th><th> Catalogo </th><th> Commenti </th></tr><tr><td> Fenilefrina HCl 2,5% </td><td> Bausch &amp; Lomb </td><td> 053-11 </td><td> Soluzione oftalmica </td></tr><tr><td> 30-Ago ga </td><td> BD </td><td> 305128 </td><td> Conica regolare </td></tr><tr><td> 10-ml Luer-Lock siringa </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 3/10-cc insulina siringa </td><td> BD </td><td> 328431 </td><td></td></tr><tr><td> Tamponi imbevuti di alcool </td><td> BD </td><td> 326895 </td><td></td></tr><tr><td> 6-pozzetti, Tissue Culture-trattati </td><td> BD </td><td> 353046 </td><td></td></tr><tr><td> Proparacaina HCl 0,5% </td><td> Maggiordomo AHS </td><td> 017239 </td><td> Soluzione oftalmica </td></tr><tr><td> Ketamina HCl 100mg/ml </td><td> Fort Dodge </td><td> 4402A </td><td></td></tr><tr><td> 10-microlitri Hamilton siringa </td><td> Hamilton Azienda </td><td> 7648-01 </td><td></td></tr><tr><td> 32-Ago ga Hamilton </td><td> Hamilton Azienda </td><td> 7803-04 </td><td> Personalizzati lunghezza - 0,5 &quot; </td></tr><tr><td> Neurobasal-A Media </td><td> Invitrogen </td><td> 10888-022 </td><td></td></tr><tr><td> B-27 Minus supplemento di vitamina A, 50X </td><td> Invitrogen </td><td> 12587-010 </td><td></td></tr><tr><td> RPMI-1640 media </td><td> Mediatech </td><td> 10-040-CV </td><td></td></tr><tr><td> Non essenziali soluzione di aminoacidi, 100X </td><td> Mediatech </td><td> 25-025-CI </td><td></td></tr><tr><td> L-Glutammina, 100X </td><td> Mediatech </td><td> 25-005-CI </td><td></td></tr><tr><td> Disposable Scalpel # 11 </td><td> Miltex </td><td> 4-411 </td><td></td></tr><tr><td> Roditori Anestesia Combo </td><td> n / a </td><td> n / a </td><td> In-house formulazione farmacia (ketamina 37,6 mg / ml, xilazina 1,92 mg / ml, acepromazina 0,38 mg / ml) </td></tr><tr><td> Fattore umano ricombinante crescita epidermico (EGF) </td><td> StemCell Technologies </td><td> 02633 </td><td> Ricostituire a 10 mg / mL di soluzione di magazzino </td></tr><tr><td> Ricombinante umano di base fattore di crescita dei fibroblasti (bFGF) </td><td> StemCell Technologies </td><td> 02634 </td><td> Ricostituire a 10 mg / mL di soluzione di magazzino </td></tr><tr><td> OMS-75 Microscopio di funzionamento </td><td> Topcon Medical Systems </td><td> OMS-75 </td><td> Questo modello è stato interrotto </td></tr><tr><td> Formalina 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

Coltura di cellule tumorali retinoblastoma nei media di cellule staminali definite dà luogo a tumorspheres primario che può essere coltivata e mantenuta solo per un tempo limitato. Queste tumorspheres colta possono presentare fenotipi cellulari notevolmente diverso rispetto al tumore originale. Dimostrazione che le cellule in coltura hanno la capacità di formare nuovi tumori è importante assicurarsi che il modello di una coltura di cellule la biologia del tumore originale. Qui vi presentiamo un protocollo per la propagazione dei tumori retinoblastoma umano in vivo utilizzando RAG2 - / - immune topi deficienti. Colta tumorspheres retinoblastoma umano di passaggio basso o cellule ottenute da appena raccolte tumori retinoblastoma umano iniettato direttamente in camera vitreale di occhi murino formare tumori entro 2-4 settimane. Questi tumori possono essere raccolte e sia ulteriori diversi passaggi negli occhi di topo in vivo o coltivate come tumorspheres in vitro. Propagazione è stato svolto con successo per almeno tre passaggi stabilendo così una continua fonte di tessuto retinoblastoma umano per ulteriori sperimentazioni. Wesley S. Bond e Lalita Wadhwa sono co-primi autori.

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

Un metodo è descritto per propagare i tumori retinoblastoma umano nei topi. Le cellule tumorali sono direttamente iniettate negli occhi di topi privi di sistema immunitario. Tumori secondari sono state stabilite con successo utilizzando sia le cellule raccolte direttamente da tumori umani e tumorspheres colta.

Istituzione e Propagazione dei Tumori retinoblastoma umani in topi privi di sistema immunitario

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

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Medicine

11.7K Views.  Baylor College of Medicine. Un metodo è descritto per propagare i tumori retinoblastoma umano nei topi. Le cellule tumorali sono direttamente iniettate negli occhi di topi privi di sistema immunitario. Tumori secondari sono state stabilite con successo utilizzando sia le cellule raccolte direttamente da tumori umani e tumorspheres colta.

Video: 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 colonscopia flessibile in topi per valutare la gravità della colite e colon-retto Tumori Utilizzo di un sistema Validated endoscopica Scoring

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

Ricerca

Medicina

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.

Induzione di Invasive Transitional cellulare Bladder Carcinoma in immunitario intatto umani MUC1 topi transgenici: un modello di sviluppo Immunoterapia

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 Imaging ottico dei tumori cerebrali e artrite Uso fluorescente SAPC-DOPS nanovesicles

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.

Femorale aspirazione del midollo osseo 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 ...

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.

Creazione anatomicamente accurati e riproducibili intracranica xenotrapianti di tumori cerebrali umani

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.

Istituzione di umano epiteliali Enteroids e Colonoids dal tessuto intero e biopsia

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.

Istituzione di un mieloma multiplo umano Xenograft modello nel pollo per studiare la crescita del tumore, invasione e l&#39;angiogenesi

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.

Induzione di aterosclerosi accelerata nei topi: il modello "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.

Caratterizzazione di cellule immunitarie nel tessuto adiposo umano mediante citometria a flusso

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.