Nous tenons à remercier Lorna Renshaw (Breast Unit Edimbourg) pour son aide précieuse dans l&#39;obtention des échantillons cliniques. Utilisation de matériel tumoral a été approuvé par le comité éthique de la recherche Lothian (08/S1101/41) et obtenu sous les auspices du Centre de médecine expérimentale du cancer de programme (Edimbourg). Ce travail est soutenu par le Conseil écossais de financement. ADL est une Mlle Margaret Butters Reekie confiance moniteur clinique.

1. Extraction du collagène des queues de rat <ol><li> Placez congelés queues de rat frais dans l&#39;éthanol à 70%. </li><li> Deglove la peau recouvrant avec un scalpel, d&#39;exposer les tendons. </li><li> Bande tendons loin de la queue et sa place dans l&#39;éthanol à 70% pour stériliser. </li><li> Peser les tendons collectées et transfert à l&#39;acide acétique (tendon 1g à 250ml d&#39;acide acétique 0,5 M). Mélanger pendant 48 h à 4 ° C. </li><li> Centrifuger le mélange d&#39;acide tendon / acide acétique à 10.000 g pendant 30 minutes et jeter le culot. </li><li> Ajouter un volume égal de 10% (p / v) de NaCl au surnageant et laisser le mélange reposer une nuit à 4 ° C. </li><li> Recueillir les riche en collagène, couche inférieure insolubles et centrifugat pour une autre 30min à 10.000 g. </li><li> Reprendre le collagène matière riche en acide acétique 0,25 M à 4 ° C et on dialyse contre 1:1000 acide acétique à 4 ° C pendant 3 jours, en changeant le tampon de dialyse deux fois par jour. </li><li> En outre stériliser la solution de collagène par centrifugation (20000 g pour 2h) et stocker à 4 ° C. </li><li> Diluer collagène tel que requis par l&#39;ajout de l&#39;acide acétique 1:1000 stériles à une concentration stock de 1mg/ml. </li></ol> 2. Création Assay 3D Le test 3D est basée sur un dosage de la lignée cellulaire, publiés précédemment 2. <ol><li> Biopsies multiples sont récoltées de patients consentants au moment de la résection chirurgicale curative pour cancer du sein invasif. </li><li> Par les yeux, diviser les noyaux en utilisant un scalpel dans 1mm 3 fragments. Trim et jeter le gras macroscopiquement distincts. </li><li> Plongez les morceaux dans les médias MEGM complète. Le tissu peut être stocké dans cet état jusqu&#39;à 1h. </li><li> Pour créer le gel de collagène, mélanger sur glace 1mg/ml collagène de queue de rat, l&#39;acide filtrée 1:1000 acétique, 10x DMEM/F12 et 0,22 M de NaOH dans un rapport de 3:5:1:1 à créer une concentration de collagène finale de 0,3 mg / ml. </li><li> Injecter 1,2 ml de mélange de collagène dans des puits individuels d&#39;une plaque de 24 puits et la placer dans une étuve à 37 ° C pendant 10min pour initier la gélification. </li><li> Après 10min, enlever la plaque de 24 puits de l&#39;incubateur. Placez les morceaux de tumeur sur le collagène de gélification et délicatement les pousser dans le centre du gel en utilisant une pointe 10 ul pipette. Retour de la plaque à 24 puits à l&#39;incubateur pendant 1 heure. </li><li> Pour les tumeurs ER +, supplément MEGM média complet avec β-estradiol à une concentration de 3.2x10 -10 M. Une fois les tests de gel ont gélifié pour 1h, 1,2 ml introduire prudemment des médias complétées dans chaque puits. La finale β-oestradiol concentration dans le dosage sera s&#39;équilibrer à 1.6x10 -10 M de récapituler les niveaux d&#39;oestrogène physiologiques dans les tissus mammaires 3. </li><li> Exécuter une pointe de pipette 10 ul autour du bord intérieur de chaque puits, libérant chaque gel circonférentiellement, permettant ainsi à chaque gel à flotter librement dans les médias. Retour de la plaque à 24 puits à l&#39;incubateur. </li><li> Bourse complété les médias deux fois par semaine jusqu&#39;à la fin de l&#39;expérience. Fin à l&#39;expérience par la suppression des médias et en ajoutant un fixateur de formol. </li></ol> 3. Coloration des anticorps du dosage dans la préparation de la tomographie optique de projection Nous avons adopté une méthode précédemment publiée pour 4 échantillons coloration entier. <ol><li> Lavez le formol échantillons fixés pendant 30min dans du PBS. Déshydrater les gels par étapes dans le méthanol dilué dans du PBS contenant 0,05% de Tween 20 (0,05% PBST): 33%, 66% et 100% pour ≥ 15min à chaque étape. </li><li> Incuber les tissus fraîchement préparés MeOH: DMSO: 30% H 2 O 2 à un ratio de 02:01:03 (15% v / v H 2 O 2) à température ambiante pendant 24 heures pour étancher l&#39;autofluorescence. </li><li> Lavez échantillons à deux reprises pour 30min dans le méthanol. Congeler les échantillons à -80 ° C à cinq reprises au moins 1h à chaque fois et revenir à température ambiante (TA) pour s&#39;assurer que les antigènes dans les parties les plus profondes du tissu sont rendus accessibles. </li><li> Réhydrater les tissus Retour à PBST 0,05% avec du méthanol comme diluant (33%, 66% et 100% pour 15min à chaque étape). </li><li> Bloquer le tissu dans une solution de blocage (10% de sérum dans le PBST 0,05%) pour 24 heures. </li><li> Incuber le tissu avec l&#39;anticorps primaire afin d&#39;identifier le contenu épithéliales, en utilisant de lapin anti-cytokératine à une concentration de 1:500 dilué dans 1,5 ml de solution de blocage contenant 5% de DMSO pour 48h. </li><li> Lavez échantillons quatre fois avec PBST 0,05% pendant 30 minutes chacun. </li><li> Incuber le tissu avec fluorescentes anticorps secondaire (1:100 chèvre anti-lapin Alexa555), dilué dans une solution de blocage 1.5ml contenant du DMSO 5% pour 48h. </li><li> Laver les échantillons à nouveau avec 0,05% PBST quatre fois pendant 30 minutes chacun. </li></ol> 4. Tomographie optique de projection La tomographie optique de projection est une technique de microscopie précédemment développés pour produire des images haute résolution en 3D de spécimens biologiques fluorescentes avec une épaisseur allant jusqu&#39;à 15 millimètres 5. <ol><li> Montez le scontenue dans un gel à 1% d&#39;agarose à faible point de fusion et déshydrater dans le méthanol à 100% pour 24 heures. Effacer le spécimen à Babb solution (01h02 alcool benzylique, le benzoate de benzyle) pour 48h et de numérisation en utilisant le scanner OPT 3001M (Bioptonics). </li><li> Les deux cibles et autofluorescence sont capturés en utilisant l&#39;ensemble Cy3 filtre (l&#39;excitateur 545nm/30 nm, émetteur 610/75 nm) et GFP1 ensemble de filtres (d&#39;excitation 425nm/40 nm, émetteur LP475 nm) respectivement. </li></ol> 5. Reconstruction 3D et la quantification <ol><li> L&#39;ensemble des projections angulaires acquises par l&#39;OPT peut être reconstituée à l&#39;aide du logiciel NRecon (SkyScan) pour créer une série de tranches de section transversale à travers l&#39;analyse d&#39;invasion. Ce logiciel est disponible au téléchargement gratuitement à <a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> . </li><li> Analyser les données reconstruites en utilisant le logiciel Volocity (PerkinElmer). Logiciel de démonstration gratuite est disponible pour téléchargement à <a href="http://www.cellularimaging.com/products/Volocity/demo/">http://www.cellularimaging.com/products/Volocity/demo/</a> . </li><li> Afin de quantifier le volume épithéliales par coloration cytokératine, fixer un seuil minimum d&#39;intensité de voxels pour réduire les interférences de la fluorescence de fond dans le canal cible. Nous avons défini ce seuil empiriquement à 9000 UA utilisant une liste de paramètres disponibles dans l&#39;application de mesure, nous avons quantifié le volume de l&#39;épithélium en continuité avec le noyau originel (délimitées dans le canal autofluorescente). Cela nous a permis de quantifier les volumes d&#39;excroissance épithéliale entre les différents échantillons tumoraux. </li></ol> 6. Les résultats représentatifs: Pendant le développement de l&#39;analyse, un total de 52 cancers du sein biopsies avec une large gamme d&#39;histopathologie ont été utilisés. Parmi ces &lt;10% (5 / 52) a omis de fournir au moins un dosage d&#39;invasion viable. L&#39;échec de dosage a été due à des bactéries soit supra-infection ou d&#39;insuffisance matérielle biopsie tumorale. Par conséquent l&#39;application du test n&#39;est pas limité à un sous-type spécifique de cancer du sein. Dosages typiques (figure 1) fixée au bout de 20 jours en culture de collagène et ensuite colorées pour la cytokératine. Logiciels Volocity est capable de reconnaître à la fois la plus grande partie épithéliale continue avec le noyau originel et les groupes détachés d&#39;envahir les cellules et quantifier chaque volume séparément. <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="Figure 1" /> Figure 1 Invasion des dosages. Après la fixation et à la quantification. Le test prend en charge les échantillons obtenus à partir de plusieurs sous-types de cancer du sein: a) ER + b) HER2 + et 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 et JF sont employés par MRC Technology, qui commercialise des scanners Bioptonics OPT. D&#39;autres auteurs déclarent aucun conflit d&#39;intérêt.

À base de collagène essais pour étudier le comportement des lignées cellulaires de cancer sont maintenant largement utilisés 6,7,8. Néanmoins, ces essais n&#39;ont pas reflété pleinement la complexité de la tumeur et son environnement. Dans cette étude, nous montrons que du matériel humain cancer du sein peut être utilisé d&#39;une manière semblable, ex vivo. Par ailleurs, l&#39;OPT est un outil utile pour caractériser l&#39;expansion 3D de cancer du sein. Nous avons constaté que la principale limitation de cette technique est la disponibilité du matériel tumoral (une seule biopsie est suffisant pour 4-6 dosages). Un autre facteur limitatif est que l&#39;invasion de structure ne survient que dans environ 40% des essais. Pour surmonter cela, nous ajoutons régulièrement de tout traitement désiré journée autour de 7 à tous les tests avec une croissance visible. L&#39;utilisation de l&#39;OPT pourrait être étendu à l&#39;avenir également de détecter les changements en vrac, ex vivo, résultant d&#39;un traitement médicamenteux. Ce pourrait à son tour de faciliter le traitement du cancer plus individualisée, indépendante des modèles animaux. Nous étudions actuellement la réponse des tumeurs ER + au tamoxifène ex vivo.

<table border="1"><tbody><tr><td> Nom de réactif </td><td> Société </td><td> No de catalogue </td></tr><tr><td> Remplissez MEGM </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-oestradiol </td><td> Sigma-Aldrich </td><td> E8875-250MG </td></tr><tr><td> Lapin anti-cytokératine </td><td> Dako </td><td> Z0622 </td></tr><tr><td> Chèvre anti-lapin Alexa555 </td><td> Invitrogen </td><td> A21422 </td></tr></tbody></table> Tous les autres réactifs standard ont été obtenus auprès de Sigma-Aldrich.

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

Le cancer du sein est une cause majeure de mortalité dans le monde occidental. Il est bien établi que la propagation du cancer du sein, d&#39;abord localement et plus tard distalement, est un facteur majeur dans le pronostic du patient. Systèmes expérimentaux de cancer du sein s&#39;appuient sur des lignées de cellules généralement tirées de tumeurs primaires ou un épanchement pleural. Deux obstacles majeurs entravent cette recherche: (i) certains sous-types connus de cancers du sein (notamment mauvais pronostic des tumeurs luminales B) ne sont pas représentés dans les collections de la ligne actuelle, (ii) l&#39;influence du microenvironnement de la tumeur n&#39;est généralement pas pris en compte. Nous démontrons une technique pour la culture primaire spécimens de cancer du sein de tous les sous-types. Ceci est réalisé en utilisant trois dimensions du système de culture (3D) dans lequel de petits morceaux de tumeur sont intégrés dans le collagène chez le rat douce, je coussins. Dans 2-3 semaines, la propagation des cellules tumorales dans le collagène et la forme des différentes structures similaires à celles observées dans tumours1 humaine. Adipocytes viables, les cellules épithéliales et les fibroblastes au sein du noyau originel étaient évidentes sur l&#39;histologie. Cellules épithéliales malignes avec une morphologie squamoid ont démontré l&#39;invasion dans le collagène environnant. Pléomorphisme nucléaire était évidente au sein de ces cellules, avec des figures de mitose et de corps apoptotiques. Nous avons utilisé la tomographie optique de projection (OPT), une technologie d&#39;imagerie 3D, afin de quantifier l&#39;ampleur de la propagation de la tumeur dans la culture. Nous avons utilisé l&#39;OPT pour mesurer le volume en vrac de la culture tumeur, un paramètre couramment mesurés durant le traitement néo-adjuvant du cancer du sein pour évaluer la réponse au traitement médicamenteux. Ici, nous présentons une occasion pour les tumeurs du sein de la culture humaine sans sous-type et de quantifier les biais de la propagation de ces ex vivo. Cette méthode pourrait être utilisée à l&#39;avenir afin de quantifier la sensibilité au médicament dans la tumeur d&#39;origine. Cela peut fournir un modèle plus prédictif que des lignées cellulaires utilisées actuellement.

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

Nous avons développé une base de collagène<em&gt; In vitro</emTest&gt; qui favorise la prolifération et l&#39;invasion à partir d&#39;échantillons de tous les sous-types de cancer du sein. Tomographie optique de projection, une technique de microscopie en trois dimensions a été utilisée pour visualiser et de quantifier expansion de la tumeur. Ce test peut être utilisé pour quantifier la réponse aux médicaments des échantillons tumoraux individuels.

Culture à long terme des spécimens humains de cancer du sein et leur analyse en utilisant la tomographie optique de projection

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 ...

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Research

JoVE Journal

Biology

14.6K vues.  University of Edinburgh. Nous avons développé une base de collagène In vitro qui favorise la prolifération et l'invasion à partir d'échantillons de tous les sous-types de cancer du sein. Tomographie optique de projection, une technique de microscopie en trois dimensions a été utilisée pour visualiser et de quantifier expansion de la tumeur. Ce test peut être utilisé pour quantifier la réponse aux médicaments des échantillons tumoraux individuels.

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Cellules d&#39;imagerie à long terme des mammifères en utilisant la microscopie à champ large

Recherche

Biologie

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.

Un neuronale et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

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.

Normalisation Né pour tomographie par fluorescence de projection optique pour l&#39;imagerie cardiaque entier

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.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

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.

Culture ex vivo des cellules humaines primaires épithéliales des trompes de Fallope

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.

À long terme des essais de toxicité létale avec le Crustacé 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.

Enrichissement de série de souches et des cellules progénitrices des spermatogonies (SSC) dans la culture de dérivation de long terme Lines adultes CSE de souris

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.

Détecter des altérations génétiques somatiques dans des échantillons de tumeurs par Exon et le séquençage massivement parallèle

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.

Multicouche de montage pour à long terme Fiche microscopie optique de poisson zèbre

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.

Isolation, transfection et culture à long terme des cardiomyocytes adultes de souris et de rats

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