Financiamento para este projeto é fornecido pela Fundação Clayton para a Investigação e da Fundação de Pesquisa Retina.

1. Preparação de tumorspheres retinoblastoma <ol><li> Obtenção de amostra de tumor retinoblastoma em um IRB-aprovado protocolo. </li><li> Prepare fresca media de células-tronco definido (Neurobasal-A mídia, 1X B-27 suplemento menos vitamina A, 1X não essenciais solução de aminoácidos, L-glutamina 1X, 20 ng / mL EGF, 10 ng / mL bFGF). </li><li> Mecanicamente desagregar o tecido tumoral com um bisturi, usando uma técnica transversal em um tecido estéril, a cultura tratados com seis bem-chapa. Imediatamente adicione 100 mL de mídia células-tronco para definir o tecido e delicadamente espalhar o tecido picada usando o bisturi. Adicionar 5 mL dos meios de comunicação de células-tronco definido para o bem. </li><li> Incubar a 37 ° C, 5% de CO 2 em uma incubadora umidificada e inspecionar diariamente. Alguns tumorspheres deve surgir a partir do tecido tumoral interrompido quase imediatamente, com um número crescente ao longo de 2-4 dias. </li><li> Semanais, tumorspheres centrifugue a 300xg por 5 minutos, ressuspender em recém-preparados media células-tronco definido, e pipeta cima e para baixo 10 vezes para interromper maior, esferas de necrose central e permitir que novas esferas saudáveis ​​a se formar. </li></ol> 2. Preparação de células tumorais para injecção <ol><li> Se injetar tumorspheres cultivadas, gentilmente esferas pipeta para um tubo de 15 mL cônico. Pipeta suavemente para cima e para baixo com uma pipeta sorológica 10-15 vezes de interromper as esferas. </li><li> Contar as células a ser injetado através de um hemocitômetro, e alíquota, pelo menos, 50 mil células por injeção em um novo tubo. Prepare-se para muitos como injeções possível com base no número de células disponíveis. </li><li> Centrifugar a 300xg por 5 minutos. Aspirar cuidadosamente o sobrenadante e ressuspender em 5 mL de PBS. Repetir a centrifugação e aspiração, e ressuspender em 10 mL PBS por injeção. </li></ol> 3. Preparação dos animais <ol><li> Usando uma seringa de insulina, administrar uma injeção intraperitoneal de cocktail anestesia roedores (ketamina 37,6 mg / mL, xilazina 1,92 mg / mL, acepromazina 0,38 mg / mL) a 1 mL por grama de peso corporal (GBW). Espere 5 minutos para que o animal se sedado. Verifique se há batimento cardíaco e colocar o animal em uma almofada aquecida. Manter animais em uma almofada aquecida em todas as vezes até a recuperação. </li><li> Após a sedação, administrar 1 gota de HCl proximetacaína no olho direito. Substituir animais na almofada aquecida e espere um minuto. </li><li> Administrar 1 gota de fenilefrina HCl para o olho direito. Substituir o animal na almofada aquecida e aguarde 5 minutos. Se a dilatação da pupila não ocorreu, re-administrar 1 gota e esperar mais 5 minutos. </li><li> Monitorar os animais em busca de sinais de movimento ou contração muscular, que pode ocorrer em&gt; 30 minutos após a sedação. Aos primeiros sinais de movimento, administrar 1 mL / GBW de cetamina HCl (diluído a 10 mg / mL em PBS) e aguarde 5 minutos. </li></ol> 4. Injeção de células tumorais <ol><li> Coloque o animal anestesiado sob o microscópio de lado, com o olho direito para cima e com a cabeça descansando sobre uma gaze e centrado para obter um reflexo vermelho do fundo do olho direito (pupila dilatada deve ser neste momento) quando observada através do microscópio . </li><li> Escorar o globo direito empurrando levemente para baixo simultaneamente com dois dedos sobre as pálpebras e mantenha esta posição de forma constante para o restante do procedimento. </li><li> Usando uma agulha estéril de calibre 30 acoplada a uma seringa Luer-lock, furar o globo lateralmente através da conjuntiva e esclera adjacente ao limbo da córnea na área da pars plana (Figura 1) e apenas através da coróide na cavidade vítrea. Retire a agulha da abertura. </li><li> Esterilizar a agulha Hamilton com um algodão embebido em álcool. Chamar a suspensão de células para dentro da seringa Hamilton e inserir a agulha na abertura até que a agulha é visualizado através do microscópio atrás da lente no vítreo perto da retina. Com a ajuda de uma segunda pessoa, enquanto segura a agulha firmemente nesta posição, o êmbolo lentamente. Remova a agulha. Se necessário, use um cotonete para absorver qualquer líquido da abertura. </li><li> Gentilmente liberar a pressão das pálpebras para fechar as pálpebras mouse. </li><li> Administrar 1 gota de hipromelose para cada olho e voltar animal para almofada aquecida para recuperação. </li></ol> 5. Monitoramento de camundongos injetados e colheita de tumores <ol><li> Diária, examine o olho injetado de leucocoria (reflexo branco papilar) e / ou distensão periocular que geralmente se torna aparente 2-4 semanas após a injecção. </li><li> Uma vez que o crescimento do tumor é detectado, a eutanásia de animais de acordo com as diretrizes institucionais. </li><li> Se o tumor do olho e são cobertos por pálpebras, use o bisturi para fazer duas incisões ortogonal à fenda palpebral e levante a retalhos cutâneos. </li><li> Sob visualização usando o microscópio estereoscópico, faça uma incisão circunferencial no limbo, eliminando a córnea ea lente, e dissecar cuidadosamente a maior parte da massa tumoral a partir de outros tecidos usando tweezers. Coloque o tumor em estéril RPMI-1640 mídia. </li><li> Use uma pinça para retirar o mundo aberto a partir do soquete. Para garantir um nervo ligado óptica para a invasão do tumor pode ser visualizado, puxe o mundo até que o nervo é exposto e usar a tesoura para cortar o nervo maior tempo possível. Coloque em formalina 10% para regular processamento para exame histológico. </li></ol> 6. Resultados representativos: Tumorspheres retinoblastoma começarão a aparecer a partir do tecido desagregados quase imediatamente como eles são liberados a partir da massa tumoral. Dentro de 2-4 dias, mais tumorspheres começará a forma e vai aumentar de tamanho. As esferas tendem a ser regular e exibem uma membrana bem definida, secundárias em torno do agregado (Figura 2). O animal normalmente apresenta-se com leucocoria dentro de 4 semanas pós-injeção (Figura 3b), seguido pelo alargamento do globo e distensão dos tecidos circundantes 5-8 semanas após a injeção que o tumor cresce (Figura 3c). <img src="/files/ftp_upload/2644/2644fig1.jpg" alt="Figura 1"/> Figura 1. Transversal diagrama do olho do rato que destaca as características referenciadas no protocolo. <img src="/files/ftp_upload/2644/2644fig2.jpg" alt="Figura 2"/> Figura 2. Cultura de células humanas in vitro retinoblastoma fotografada em um 4x), e b) ampliação da objetiva 10x. Retinoblastoma células do tumor primário produzir tumorspheres com uma forma regular esférica e uma membrana externa crisp. Barras de escala representam a) 500 mm, e b) M 200. <img src="/files/ftp_upload/2644/2644fig3.jpg" alt="Figura 3"/> Figura 3 Eye of RAG2 -. / - Mostrando um rato características) normal, b) leucocoria indicativo de uma massa tumoral na cavidade vítrea, e c) uma grande massa tumoral preenchendo o mundo com distensão periocular associados, hemorragia intra-ocular.

<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>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pelo Colégio Baylor de Medicina IACUC Comitê.

A técnica descrita a seguir facilita a propagação de tumores retinoblastoma no seu meio, intraocular intravítrea. A técnica de injeção intra-ocular tem sido usado no passado para criar tumores a partir de linhas de células derivadas de retinoblastoma 1, bem como para entregar vetores virais para terapia genética intra-ocular 2,3. Esta técnica já foi utilizada com sucesso para produzir tumores retinoblastoma humano por injeção direta de células do tumor primário e injeção de tumorspheres bem como a propagação de série de tumores xenoenxerto. Evidência visível de formação de tumores (geralmente leucocoria) é geralmente observado pela primeira vez dentro de 4 semanas, após o que a hemorragia intra-ocular e distensão do globo e / ou tecidos que circundam a órbita desenvolver dentro de 5-8 semanas. Uma minoria de camundongos injetados ferir o olho injetado, levando ao encerramento definitivo das pálpebras. Nestes casos, a distensão é o único sinal de formação de tumores. Estabelecimento de tumores xenoenxerto murino não foi bem sucedido com todos os tumores retinoblastoma humano, embora a propagação de tumores xenoenxerto estabelecido é altamente bem sucedido. Esta observação sugere que certas características do tumor primário, como a invasão e nível de diferenciação ou algum outro fator desconhecido, pode estar influenciando a capacidade de estes tumores de persistir no ambiente ocular murino. A quantidade de tecido que podem ser adquiridos a partir de um tumor retinoblastoma humano é muito pequeno, e há limitações significativas sobre a capacidade de células cultura humana retinoblastoma in vitro, tais como tempo de vida limitado, a perda de histologia do tumor sólido e mudanças no fenótipo celular. Este protocolo fornece uma maneira relativamente simples de propagar o tumor humano e estabelecer um modelo murino da doença humana. Isso permite que mais in vitro e em experimentação in vivo de biologia do retinoblastoma e de manutenção mais longos do lado de fora do tumor do paciente.

<table border="1"><tbody><tr><th> Nome </th><th> Companhia </th><th> Catalogo </th><th> Comentários </th></tr><tr><td> Fenilefrina HCl 2,5% </td><td> Bausch &amp; Lomb </td><td> 053-11 </td><td> Solução oftálmica </td></tr><tr><td> 30-ga Agulha </td><td> BD </td><td> 305128 </td><td> Bisel regulares </td></tr><tr><td> 10 ml Seringa Luer-Lock </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 3/10-cc seringa de insulina </td><td> BD </td><td> 328431 </td><td></td></tr><tr><td> Compressas com álcool </td><td> BD </td><td> 326895 </td><td></td></tr><tr><td> 6-Bom Prato Tecido, Cultura-Tratada </td><td> BD </td><td> 353046 </td><td></td></tr><tr><td> Proximetacaína HCl 0,5% </td><td> Butler AHS </td><td> 017239 </td><td> Solução oftálmica </td></tr><tr><td> Ketamina HCl 100mg/ml </td><td> Fort Dodge </td><td> 4402A </td><td></td></tr><tr><td> 10 mL Hamilton Seringa </td><td> Empresa Hamilton </td><td> 7648-01 </td><td></td></tr><tr><td> 32-ga Hamilton Agulha </td><td> Empresa Hamilton </td><td> 7803-04 </td><td> Comprimento personalizado - 0.5 &quot; </td></tr><tr><td> Neurobasal-A Mídia </td><td> Invitrogen </td><td> 10888-022 </td><td></td></tr><tr><td> B-27 Suplemento Minus vitamina A, 50X </td><td> Invitrogen </td><td> 12587-010 </td><td></td></tr><tr><td> RPMI-1640 Mídia </td><td> Mediatech </td><td> 10-040-CV </td><td></td></tr><tr><td> Não essenciais solução de aminoácidos, 100X </td><td> Mediatech </td><td> 25-025-CI </td><td></td></tr><tr><td> L-Glutamina, 100X </td><td> Mediatech </td><td> 25-005-CI </td><td></td></tr><tr><td> Descartáveis ​​# 11 Scalpel </td><td> Miltex </td><td> 4-411 </td><td></td></tr><tr><td> Combo Anestesia roedores </td><td> n / a </td><td> n / a </td><td> Na casa de formulação de farmácia (ketamina 37,6 mg / mL, xilazina 1,92 mg / mL, acepromazina 0,38 mg / mL) </td></tr><tr><td> Recombinante Fator de crescimento epidérmico humano (EGF) </td><td> StemCell Technologies </td><td> 02633 </td><td> Reconstituir a 10 mg / mL solução estoque </td></tr><tr><td> Fator de Crescimento Humano Recombinante fibroblasto básico (bFGF) </td><td> StemCell Technologies </td><td> 02634 </td><td> Reconstituir a 10 mg / mL solução estoque </td></tr><tr><td> OMS-75 Microscópio Operação </td><td> Topcon Medical Systems </td><td> OMS-75 </td><td> Este modelo foi descontinuado </td></tr><tr><td> Formalina a 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

A cultura de células de tumor retinoblastoma nos meios de comunicação célula-tronco dá origem definida para tumorspheres primária que pode ser cultivado e mantido apenas por um período limitado. Estes tumorspheres cultivadas podem apresentar marcadamente diferentes fenótipos celulares quando comparado aos tumores original. Demonstração de que as células cultivadas têm a capacidade de formar novos tumores é importante para garantir que o modelo células cultivadas a biologia do tumor original. Aqui apresentamos um protocolo para a propagação de tumores retinoblastoma humano in vivo utilizando RAG2 - / - ratos imunológico deficiente. Cultivadas tumorspheres retinoblastoma humano de passagem baixa ou células obtidas de recém-colhido tumores retinoblastoma humano injetado diretamente na cavidade vítrea de olhos murino formar tumores dentro de 2-4 semanas. Estes tumores podem ser colhidas e quer ainda várias passagens nos olhos murino in vivo ou crescido como tumorspheres in vitro. Propagação foi realizado com sucesso há pelo menos três passagens estabelecendo assim uma fonte contínua de tecido retinoblastoma humano para fazer novas experiências. Wesley S. Bond e Lalita Wadhwa são co-autores primeiro.

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

Um método é descrito para propagar tumores retinoblastoma humano em camundongos. Células tumorais são diretamente injetadas nos olhos de ratos imune deficiente. Tumores secundários foram estabelecidas com sucesso usando as duas células diretamente colhidas a partir de tumores humanos e tumorspheres cultivadas.

Estabelecimento e propagação de Tumores Humanos em Retinoblastoma Imune camundongos deficientes

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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Research

JoVE Journal

Medicine

11.7K visualizações.  Baylor College of Medicine. Um método é descrito para propagar tumores retinoblastoma humano em camundongos. Células tumorais são diretamente injetadas nos olhos de ratos imune deficiente. Tumores secundários foram estabelecidas com sucesso usando as duas células diretamente colhidas a partir de tumores humanos e tumorspheres cultivadas.

Vídeo: 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.

A colonoscopia flexível em camundongos para avaliar a gravidade da colite e colorretal Tumores Usando um sistema validado endoscópica Scoring

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

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.

Indução de carcinoma da bexiga invasivo de células transicionais em imunológico intacto Humanos MUC1 Ratos transgênicos: um modelo de desenvolvimento Imunoterapia

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 Imagem Óptico de tumores cerebrais e artrite Usando fluorescente SAPC-DOPS nanovesículas

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.

Femoral Medula Óssea aspiração Live Ratos

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.

Criando anatomicamente precisos e reprodutíveis intracraniana Xenoenxertos de tumores cerebrais humanos

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.

Estabelecimento de epiteliais humanas Enteroids e Colonoids de tecido inteiro e biópsia

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.

Criação de um Modelo de Xenoenxerto Humano mieloma múltiplo na galinha para estudar o crescimento tumoral, invasão e angiogênese

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.

Indução de Aterosclerose Acelerada em Camundongos: O Modelo de "Lesão por Fio"

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.

Caracterização de células imunes no tecido adiposo humano usando citometria de fluxo

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.