Agradecemos a Lorna Renshaw (Edinburgh Breast Unit) para sua inestimável assistência na obtenção de amostras clínicas. Uso de material tumoral foi aprovado pelo Comitê de Ética de Pesquisa Lothian (08/S1101/41) e obtido sob os auspícios do Centro Experimental Cancer Medicine programa (Edinburgh). Este trabalho é apoiado pelo Conselho de Financiamento escocês. ADL é uma senhorita Margaret Butters Reekie companheiros Confiança clínico.

1. Extração de colágeno de Tails Rat <ol><li> Lugar fresco congelado rabos de rato em etanol 70%. </li><li> Deglove a pele sobrejacente com um bisturi expondo tendões. </li><li> Faixa de tendões longe de cauda e colocar em etanol 70% para esterilizar. </li><li> Pesar os tendões coletados e transferidos para o ácido acético (1g tendão ao ácido acético 0,5 M 250ml). Mix de 48hr a 4 ° C. </li><li> Centrifugar a mistura de ácido tendão / acético a 10.000 g por 30min e descartar o pellet. </li><li> Adicionar um volume igual a 10% (w / v) NaCl para o sobrenadante e deixar a mistura repousar uma noite a 4 ° C. </li><li> Recolher as ricas em colágeno, camada inferior insolúveis e centrifugado para um mais 30min a 10.000 g. </li><li> Ressuspender o material rico em colágeno em ácido acético 0,25 m a 4 ° C e dialyse contra 1:1000 ácido acético a 4 ° C por 3 dias, mudando o tampão de diálise duas vezes por dia. </li><li> Além disso esterilizar a solução de colágeno por centrifugação (20.000 g para 2hr) e armazenar a 4 ° C. </li><li> Diluir colágeno conforme requerido, com a adição de ácido acético 1:1000 estéril a uma concentração de ações de 1mg/ml. </li></ol> 2. 3D Criação de Ensaio O ensaio 3D é baseado em um ensaio de linha celular, publicado anteriormente 2. <ol><li> Biópsias múltiplas são colhidas consentindo pacientes no momento da ressecção cirúrgica curativa para o câncer de mama invasivo. </li><li> Por olho, divide os núcleos com um bisturi em um milímetro três fragmentos. Guarnição e descartar gordura macroscopicamente distintos. </li><li> Mergulhe os fragmentos em MEGM mídia completa. O tecido pode ser armazenado neste estado por até 1h. </li><li> Para criar o gel de colágeno mix, no gelo colágeno da cauda de rato 1mg/ml, 1:1000 ácido acético filtrada, DMEM/F12 10x e 0,22 m NaOH em uma proporção de 3:5:1:1 para criar uma concentração de colágeno final de 0,3 mg / ml. </li><li> Injetar 1,2 ml da mistura de colágeno em poços individuais de uma placa de 24 poços e coloque em uma incubadora a 37 ° C por 10 min para iniciar gelificação. </li><li> Após 10min, retire a placa de 24 poços da incubadora. Coloque os pedaços de tumor para o colágeno gelificação e suavemente empurrá-los para o centro do gel usando uma ponteira 10μl. Devolver a placa de 24 poços para a incubadora de 1hr. </li><li> Para ER + tumores, suplemento MEGM media completo com β-estradiol a uma concentração de 3.2x10 -10 M. Uma vez que os ensaios de gel têm gel para 1hr, cuidadosamente introduzir 1,2 ml do meio suplementado em cada poço. A concentração de β-estradiol final no ensaio vai para equilibriate 1.6x10 M -10 a recapitular os níveis de estrogênio fisiológicas encontradas no tecido mamário 3. </li><li> Executar uma ponteira 10μl em torno da borda interna de cada bem, liberando cada gel circunferencialmente, permitindo assim que cada gel para flutuar livremente na mídia. Devolver a placa de 24 poços para a incubadora. </li><li> Troca complementada media duas vezes por semana até o término do experimento. Rescindir o experimento através da remoção de mídia e adicionando um fixador formalina. </li></ol> 3. Coloração de anticorpos de Ensaio em preparação para Tomografia projeção óptica Adotamos um método previamente publicados por 4 toda amostra de coloração. <ol><li> Lavar formol espécimes fixados por 30min em PBS. Desidratar o gel stepwise em metanol diluído em PBS contendo 0,05% Tween 20 (PBST 0,05%): 33%, 66% e 100% para ≥ 15min em cada etapa. </li><li> Incubar o tecido em MeOH preparados na hora: DMSO: 30% H 2 O 2 em uma proporção de 02:01:03 (15% v / v H 2 O 2) em temperatura ambiente por 24 horas para saciar a autofluorescência. </li><li> Lavar as amostras duas vezes por 30min em metanol. Congelar as amostras a -80 ° C cinco vezes por pelo menos 1 hora de cada vez e de volta à temperatura ambiente (RT) para garantir que os antígenos nas partes mais profundas do tecido são tornados acessíveis. </li><li> Reidratar o tecido de volta para PBST 0,05% com metanol como solvente (33%, 66% e 100% por 15 min em cada etapa). </li><li> Bloquear o tecido em solução de bloqueio (10% de soro em PBST 0,05%) para 24 horas. </li><li> Incubar o tecido com o anticorpo primário para identificar o conteúdo epiteliais, usando de coelho anti-citoqueratina na concentração de 1:500 diluídos em 1,5 ml de solução bloqueadora contendo DMSO 5% para 48hr. </li><li> Lavar as amostras quatro vezes com PBST 0,05% para cada 30min. </li><li> Incubar o tecido com anticorpo fluorescente secundário (1:100 cabra anti-coelho Alexa555), diluído em solução de bloqueio de 1,5 ml contendo DMSO 5% para 48hr. </li><li> Lavar as amostras novamente com PBST 0,05% quatro vezes por 30min cada. </li></ol> 4. Tomografia projeção óptica Tomografia projeção óptica é uma técnica de microscopia previamente desenvolvido para produzir imagens de alta resolução em 3D de espécimes biológicos fluorescentes com uma espessura de até 15 milímetros 5. <ol><li> Monte o sgel sustentado em 1% agarose baixo ponto de fusão e desidratação em metanol 100% para 24h. Limpar o espécime em Babb solução (1:2 álcool benzílico, Benzoato de benzilo) para 48hr e digitalizar usando o OPT Scanner 3001M (Bioptonics). </li><li> Alvo e autofluorescência são capturados usando o conjunto de Cy3 filtro (excitador 545nm/30 nm, emissor 610/75 nm) e GFP1 conjunto de filtros (excitador 425nm/40 nm, emissor LP475 nm), respectivamente. </li></ol> 5. Reconstrução e quantificação em 3D <ol><li> O conjunto de projeções angular adquirida pela OPT pode ser reconstruído usando NRecon software (SkyScan) para criar uma série de fatias de seção transversal através do ensaio de invasão. Este software está disponível para download gratuitamente no <a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> . </li><li> Analisar os dados reconstruídos usando software Volocity (PerkinElmer). Software de demonstração gratuita está disponível para download em <a href="http://www.cellularimaging.com/products/Volocity/demo/">http://www.cellularimaging.com/products/Volocity/demo/</a> . </li><li> Para quantificar o volume epiteliais através da coloração citoqueratina, definir um limite de intensidade mínima voxel para reduzir a interferência da fluorescência de fundo no canal alvo. Definimos esse limite empiricamente em 9000 au Usando uma lista de parâmetros disponíveis no aplicativo de medição, nós quantificamos o volume de epitélio em continuidade com o núcleo original (demarcadas no canal autofluorescent). Isso nos permitiu quantificar volumes de crescimento epitelial entre diferentes amostras de tumor. </li></ol> 6. Resultados representativos: Durante o desenvolvimento do ensaio, um total de 52 biópsias de câncer de mama com uma ampla gama de estudos histopatológicos, foram utilizados. Destes &lt;10% (5 / 52) não conseguiram fornecer pelo menos um ensaio de invasão viável. Falha ensaio foi devido a qualquer bacteriana supra-infecção ou insuficiente tumor material de biópsia. Portanto, aplicação do ensaio não está restrito a um subtipo específico de cancro da mama. Ensaios típico (figura 1) fixo após 20 dias na cultura de colágeno e, posteriormente, coradas para citoqueratina. Volocity software é capaz de reconhecer tanto a granel, a epitelial contínua com o núcleo original e grupos destacados de invadir células e quantificar cada volume separadamente. <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="Figura 1" /> Figura 1. Ensaios Invasion seguintes fixação e quantificação. O ensaio suporta amostras obtidas a partir de múltiplos subtipos de câncer de mama: a) ER + b) HER2 + e c) ER-.

<ol>
	<li>Lopez-Garcia, M. A., Geyer, F. C., Lacroix-Triki, M., Marchio, C., Reis-Filho, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+precursors+revisited:+molecular+features+and+progression+pathways.+Histopathology.">Breast cancer precursors revisited: molecular features and progression pathways. Histopathology.</a> 57, 171-192 (2010).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J Cell Biol. 185, 11-19 (2009).</li><li>Dabrosin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+extracellular+local+levels+of+estradiol+in+normal+breast+in+vivo+during+the+luteal+phase+of+the+menstrual+cycle.">Increased extracellular local levels of estradiol in normal breast in vivo during the luteal phase of the menstrual cycle.</a> J Endocrinol. 187, 103-108 (2005).</li><li>Alanentalo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+molecular+imaging+and+3D+quantification+within+adult+mouse+organs.">Tomographic molecular imaging and 3D quantification within adult mouse organs.</a> Nat Methods. 4, 31-33 (2007).</li><li>Sharpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Hidalgo-Carcedo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+requires+suppression+of+actomyosin+at+cell-cell+contacts+mediated+by+DDR1+and+the+cell+polarity+regulators+Par3+and+Par6.">Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6.</a> Nat Cell Biol. 13, 49-58 (2011).</li><li>Katz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+on+the+HER2+amplicon,+C35,+is+an+oncogene+in+breast+cancer+whose+actions+are+prevented+by+inhibition+of+Syk.">A gene on the HER2 amplicon, C35, is an oncogene in breast cancer whose actions are prevented by inhibition of Syk.</a> Br J Cancer. 103, 401-410 (2010).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li></ol>

SEW e JF são empregados por MRC Tecnologia, que está comercializando scanners Bioptonics OPT. Outros autores declaram que não há conflito de interesses.

À base de colágeno ensaios para estudar o comportamento de linhagens de células de câncer são hoje amplamente utilizadas 6,7,8. No entanto, estas análises não têm refletido na complexidade do total do tumor e seu ambiente. Neste estudo, nós mostramos que materiais câncer de mama humano pode ser usado de uma maneira similar, ex vivo. Além disso, OPT é uma ferramenta útil para caracterizar a expansão 3D de câncer de mama. Descobrimos que a principal limitação desta técnica é a disponibilidade de materiais tumor (um único núcleo biópsia é suficiente para 4-6 ensaios). Outro fator limitante é que a invasão estrutural ocorre apenas em cerca de 40% dos ensaios. Para superar isso, nós regularmente adicionar qualquer tipo de tratamento desejado por volta do dia 7 para todos os ensaios com o crescimento visível. O uso do OPT poderia ser estendido no futuro para também detectar alterações em massa, ex vivo, resultantes do tratamento da toxicodependência. Este por sua vez, poderia facilitar o tratamento do câncer mais individualizada, independente de modelos animais. Estamos a explorar a resposta de tumores ER + ao tamoxifeno ex vivo.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> No. de catálogo </td></tr><tr><td> MEGM Completo </td><td> Lonza </td><td> CC-3150 </td></tr><tr><td> DMEM/F12 </td><td> Sigma-Aldrich </td><td> D8900 10X1L </td></tr><tr><td> β-estradiol </td><td> Sigma-Aldrich </td><td> E8875-250mg </td></tr><tr><td> Coelho anti-citoqueratina </td><td> Dako </td><td> Z0622 </td></tr><tr><td> Cabra anti-coelho Alexa555 </td><td> Invitrogen </td><td> A21422 </td></tr></tbody></table> Todos os outros reagentes padrão foram obtidos da Sigma-Aldrich.

long-term culture of human breast cancer specimens and their analysis using optical projection tomography

Câncer de mama é uma das principais causas de mortalidade no mundo ocidental. Está bem estabelecido que a propagação do câncer de mama, primeiro localmente e, mais tarde distalmente, é um fator importante no prognóstico do paciente. Sistemas experimentais de câncer de mama dependem de linhas de células normalmente derivadas de tumores primários ou derrame pleural. Dois grandes obstáculos dificultam esta pesquisa: (i) alguns conhecidos sub-tipos de cânceres de mama (principalmente pobres prognóstico tumores luminal B) não estão representados em colecções linha atual, (ii) a influência do microambiente do tumor geralmente não é tido em conta. Nós demonstramos uma técnica para a cultura amostras de câncer de mama primário de todos os tipos de sub-. Isto é conseguido usando sistema tridimensional de cultura (3D) em que pequenos pedaços de tumor são incorporados no colágeno de ratos macio eu almofadas. Dentro de 2-3 semanas, as células cancerígenas se espalham para o colágeno e formam várias estruturas semelhantes aos observados em tumours1 humana. Adipócitos viável, células epiteliais e fibroblastos dentro do núcleo original eram evidentes na histologia. Células epiteliais malignas com morfologia squamoid foram demonstradas em invadir o colágeno circundante. Pleomorfismo nuclear era evidente dentro dessas células, juntamente com figuras de mitose e corpos apoptóticos. Nós empregamos Tomografia projeção óptica (OPT), uma tecnologia de imagem 3D, a fim de quantificar o grau de disseminação do tumor em cultura. Temos usado OPT para medir o volume maior da cultura tumor, um parâmetro rotineiramente medidas durante o tratamento neo-adjuvante de pacientes com câncer de mama para avaliar a resposta à terapia medicamentosa. Aqui, apresentamos uma oportunidade de tumores de mama humano sem cultura sub-tipo de preconceito e quantificar a propagação dos ex vivo. Este método poderia ser usado no futuro para quantificar a sensibilidade da droga no tumor original. Isso pode fornecer um modelo mais preditivo do que linhas celulares usados ​​atualmente.

Watch this Scientific Journal Video about Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography at JoVE.com

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Nós desenvolvemos um à base de colágeno<em&gt; In vitro</em&gt; Ensaio que promove a proliferação e invasão a partir de amostras de todos os subtipos de câncer de mama. Tomografia projeção óptica, uma técnica de microscopia tridimensional foi utilizado para visualizar e quantificar a expansão do tumor. Este ensaio poderá ser utilizada para quantificar a resposta à droga das amostras de tumor individual.

Longo prazo Cultura de Amostras de mama Humano do Câncer e sua análise por meio de tomografia óptica de Projeção

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

long-term-culture-human-breast-cancer-specimens-their-analysis-using

Research

JoVE Journal

Biology

14.6K visualizações.  University of Edinburgh. Nós desenvolvemos um à base de colágeno In vitro Ensaio que promove a proliferação e invasão a partir de amostras de todos os subtipos de câncer de mama. Tomografia projeção óptica, uma técnica de microscopia tridimensional foi utilizado para visualizar e quantificar a expansão do tumor. Este ensaio poderá ser utilizada para quantificar a resposta à droga das amostras de tumor individual.

Vídeo: Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Longo prazo células de mamíferos utilizando imagens do Largo-Campo Microscopia

Biologia

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

Normalização nascido para Tomografia Projeção de fluorescência óptica para imagens de todo o coração

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo de Cultura Primária células epiteliais trompa de Falópio

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Enriquecimento de série-tronco de esperma e células progenitoras (SSC) em Cultura para Derivação de longo prazo Adultos Linhas rato SSC

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Detectando somáticas Genéticos Alterações em amostras de tumor por Exon Captura e Seqüenciamento Massively Parallel

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Multicamadas de montagem para longo prazo Microscopia de Luz Folha de Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Isolamento, Transfecção e Cultura de Longo Prazo de Cardiomiócitos de Ratos Adultos e Ratos

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...