Ringraziamo Lorna Renshaw (Breast Unit di Edimburgo) per la sua preziosa assistenza per ottenere campioni clinici. Utilizzo di materiale tumorale è stato approvato dal Comitato Etico di ricerca Lothian (08/S1101/41) e ottenuto sotto l&#39;egida del Centro di Medicina Sperimentale Cancer programma (Edimburgo). Questo lavoro è supportato dal Consiglio scozzese finanziamento. ADL è una signorina Margaret Butters Reekie compagni Fiducia clinica.

1. Estrarre collagene da Tails Rat <ol><li> Luogo fresco congelato code di ratto in etanolo al 70%. </li><li> Deglove la cute sovrastante i tendini con un bisturi esporre. </li><li> Striscia tendini dalla coda e il luogo in etanolo al 70% per sterilizzare. </li><li> Pesare i tendini raccolta e trasferimento in acido acetico (tendine 1g per 250 ml di acido acetico 0,5 M). Mescolare per 48 ore a 4 ° C. </li><li> Centrifugare il mix acido tendine / acetico a 10.000 g per 30 minuti e scartare il pellet. </li><li> Aggiungere un volume equivalente del 10% (w / v) di NaCl il supernatante e lasciare il mix riposare per una notte a 4 ° C. </li><li> Raccogliere il collagene ricchi, strato insolubile fondo e centrifugato per un ulteriore 30 min a 10.000 g </li><li> Risospendere il collagene materiale ricco in acido acetico 0.25M a 4 ° C e dializzato contro 1:1000 acido acetico al 4 ° C per 3 giorni, cambiando il buffer di dialisi due volte al giorno. </li><li> Sterilizzare ulteriormente la soluzione di collagene per centrifugazione (20.000 g per 2 ore) e conservare a 4 ° C. </li><li> Diluire collagene come richiesto con l&#39;aggiunta di acido sterile 1:1000 acetico ad una concentrazione stock di 1mg/ml. </li></ol> 2. Saggio di creazione 3D L&#39;analisi 3D è basato su una linea cellulare saggio, pubblicato in precedenza 2. <ol><li> Biopsie multiple core vengono raccolte da pazienti consenzienti al momento della resezione chirurgica curativa per il tumore mammario invasivo. </li><li> Ad occhio, dividere i nuclei con un bisturi in 1 millimetro 3 frammenti. Tagliare ed eliminare il grasso macroscopicamente distinte. </li><li> Immergere i frammenti in MEGM mezzi di comunicazione completa. Il tessuto può essere immagazzinato in questo stato per un massimo di 1 ora. </li><li> Per creare il gel di collagene, mix sul collagene ghiaccio coda di topo 1mg/ml, 1:1000 filtrato acido acetico, 10x DMEM/F12 e NaOH 0.22M in un rapporto di 3:5:1:1 per creare una concentrazione finale di 0,3 collagene mg / ml. </li><li> Iniettare 1,2 ml di miscela di collagene nei singoli pozzetti di una piastra ben 24 e riporre in un incubatore a 37 ° C per 10 minuti per iniziare gelificante. </li><li> Dopo 10 minuti, rimuovere la piastra ben 24 dal termostato. Mettere i pezzi tumore al collagene gelificazione e delicatamente spingere al centro del gel con una punta 10μl pipetta. Ritorna il piatto ben 24 per l&#39;incubatore per 1 ora. </li><li> Per i tumori ER +, supplemento MEGM mezzi Completo di β-estradiolo a una concentrazione di 3.2x10 -10 M. Una volta che i saggi di gel hanno gelificato per 1 ora, con attenzione introdurre 1,2 mL del supporto integrato in ciascun pozzetto. La finale β-estradiolo concentrazione all&#39;interno del saggio sarà equilibriate di 1.6x10 -10 m di ricapitolare i livelli di estrogeni fisiologici trovato nel tessuto mammario 3. </li><li> Eseguire una punta 10μl pipetta attorno al bordo interno di ogni bene, rilasciando ogni gel circonferenza in modo che ciascuno gel di galleggiare liberamente all&#39;interno dei media. Ritorna il piatto ben 24 per l&#39;incubatrice. </li><li> Scambio integrato dei media due volte alla settimana fino al termine dell&#39;esperimento. Interrompere l&#39;esperimento, eliminando i media e l&#39;aggiunta di un fissativo formalina. </li></ol> 3. La colorazione degli anticorpi del saggio in preparazione per la Tomografia Ottica di proiezione Abbiamo adottato un metodo precedentemente pubblicato per 4 intera colorazione del campione. <ol><li> Lavare campioni fissati in formalina per 30 min in PBS. Disidratare i gel graduale in metanolo diluito in PBS contenente 0,05% di Tween 20 (0,05% PBST): 33%, 66% e 100% per ≥ 15 minuti ad ogni passo. </li><li> Incubare il tessuto in appena preparato MeOH: DMSO: 30% H 2 O 2 ad un rapporto di 2:01:03 (15% v / v H 2 O 2) a temperatura ambiente per 24 ore per spegnere l&#39;autofluorescenza. </li><li> Lavare i campioni due volte per 30 minuti in metanolo. Congelare i campioni a -80 ° C cinque volte per almeno 1 ora ogni volta e torna a temperatura ambiente (RT) per assicurare che gli antigeni nelle parti più profonde del tessuto sono resi accessibili. </li><li> Reidratare il tessuto torna al 0,05% PBST con metanolo come diluente (33%, 66% e 100% per 15 minuti ad ogni passo). </li><li> Bloccare il tessuto nel bloccare soluzione (10% di siero in PBST 0,05%) per 24 ore. </li><li> Incubare il tessuto con l&#39;anticorpo primario per identificare il contenuto epiteliali, utilizzando coniglio anti-citocheratina ad una concentrazione di 1:500 diluito in 1,5 ml di soluzione bloccante contenente il 5% DMSO per 48 ore. </li><li> Lavare i campioni quattro volte con PBST 0,05% per ogni 30 min. </li><li> Incubare il tessuto fluorescente con anticorpo secondario (1:100 capra anti-coniglio Alexa555), diluito in 1,5 ml soluzione bloccante contenente il 5% DMSO per 48 ore. </li><li> Lavare i campioni di nuovo con 0,05% PBST quattro volte per 30 minuti ciascuno. </li></ol> 4. Proiezione Tomografia ottica Tomografia ottica di proiezione è una tecnica di microscopia precedentemente sviluppato per produrre immagini 3D ad alta risoluzione di campioni biologici fluorescente con uno spessore fino a 15 millimetri 5. <ol><li> Montare il sgel mantenuto in 1% di agarosio a basso punto di fusione e disidratare in 100% di metanolo per 24 ore. Cancella il campione in BABB soluzione (1:2 alcool benzilico, benzil benzoato) per 48 ore e la scansione con lo scanner OPT 3001M (Bioptonics). </li><li> Bersaglio e autofluorescenza vengono catturati utilizzando il Cy3 set di filtri (eccitatore 545nm/30 nm, emettitore 610/75 nm) e GFP1 set di filtri (eccitatore 425nm/40 nm, emettitore LP475 nm) rispettivamente. </li></ol> 5. Ricostruzione e quantificazione 3D <ol><li> L&#39;insieme di proiezioni angolari acquisita da OPT può essere ricostruita utilizzando NRecon software (SkyScan) per creare una serie di fette di sezione trasversale attraverso l&#39;analisi invasione. Questo software è disponibile per il download gratuito a <a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> . </li><li> Analizzare i dati ricostruiti utilizzando un software Volocity (PerkinElmer). Software dimostrativo gratuito è disponibile per il download all&#39;indirizzo <a href="http://www.cellularimaging.com/products/Volocity/demo/">http://www.cellularimaging.com/products/Volocity/demo/</a> . </li><li> Al fine di quantificare il volume epiteliali dalla colorazione citocheratina, impostare una soglia minima di intensità voxel per ridurre le interferenze da fluorescenza di fondo nel canale di destinazione. Abbiamo definito questa soglia empiricamente a 9000 au Utilizzando un elenco dei parametri disponibili nella richiesta di misura, abbiamo quantificato il volume di epitelio in continuità con il nucleo originario (delimitata nel canale autofluorescenti). Questo ci ha permesso di quantificare i volumi di escrescenza epiteliali tra le diverse campioni di tumore. </li></ol> 6. Rappresentante dei risultati: Durante lo sviluppo del test, un totale di 52 campioni di tumore della mammella biopsia con una vasta gamma di istopatologia sono stati utilizzati. Di questi &lt;10% (5 / 52) non è riuscito a fornire almeno un test valido invasione. Fallimento del test è dovuto a uno sovra-infezione batterica o insufficiente materiale tumorale biopsia. Pertanto l&#39;applicazione del test non è limitato a uno specifico sottotipo di tumore al seno. Saggi tipici (figura 1) fissato dopo 20 giorni in cultura collagene e successivamente colorati per citocheratina. Il software Volocity è in grado di riconoscere sia il grosso epiteliale continuo con il nucleo originario e gruppi distaccata di invadere le cellule e quantificare ogni volume separatamente. <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="Figura 1" /> Figura 1. Saggi Invasion dopo la fissazione e la quantificazione. Il saggio sostiene campioni ottenuti da più sottotipi cancro al seno: 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 sono dipendenti di MRC Technology, che sta commercializzando Bioptonics scanner OPT. Altri autori dichiarano assenza di conflitto di interesse.

A base di collagene saggi per studiare il comportamento delle linee di cellule tumorali sono ora ampiamente utilizzate 6,7,8. Tuttavia, queste analisi non hanno riflesso in pieno la complessità del tumore e del suo ambiente. In questo studio, abbiamo dimostrato che il cancro al seno umano i materiali possono essere usati in modo simile, ex vivo. Inoltre, OPT è un utile strumento per caratterizzare l&#39;espansione 3D di cancro al seno. Abbiamo scoperto che il limite principale di questa tecnica è la disponibilità di materiali tumore (un unico nucleo biopsia è sufficiente per 4-6 test). Un altro fattore limitante è che l&#39;invasione strutturale si verifica solo nel 40% dei test. Per ovviare a questo, abbiamo regolarmente aggiungere qualsiasi trattamento desiderato intorno al giorno 7 per tutti i test con una crescita visibile. L&#39;uso di OPT potrebbe essere estesa in futuro anche per rilevare i cambiamenti di massa, ex vivo, derivante dalla terapia farmacologica. Ciò a sua volta potrebbe facilitare la terapia del cancro più individualizzata, indipendente di modelli animali. Stiamo attualmente valutando la risposta dei tumori ER + a tamoxifene ex vivo.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Catalogo No. </td></tr><tr><td> Completa 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-estradiolo </td><td> Sigma-Aldrich </td><td> E8875-250mg </td></tr><tr><td> Coniglio anti-citocheratina </td><td> Dako </td><td> Z0622 </td></tr><tr><td> Capra anti-coniglio Alexa555 </td><td> Invitrogen </td><td> A21422 </td></tr></tbody></table> Tutti gli altri reagenti di serie sono stati ottenuti da Sigma-Aldrich.

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

Il cancro al seno è una delle principali cause di mortalità nel mondo occidentale. E &#39;ben noto che la diffusione del tumore al seno, prima localmente e poi distalmente, è un fattore importante nella prognosi dei pazienti. Sistemi sperimentali di tumore al seno si basano su linee cellulari derivate da tumori di solito primaria o versamento pleurico. Due gli ostacoli principali ostacolare questa ricerca: (i) alcune note sotto-tipi di tumori al seno (in particolare scarsa prognosi dei tumori luminale B) non sono rappresentati all&#39;interno di raccolte di linea corrente, (ii) l&#39;influenza del microambiente tumorale di solito non è presa in considerazione. Dimostriamo una tecnica per i campioni del cancro al seno primario cultura di tutti i sub-tipi. Questo è ottenuto utilizzando tridimensionale (3D) sistema di coltura in cui sono inserite piccole parti di tumore nel ratto collagene I cuscini morbidi. Entro 2-3 settimane, le cellule tumorali diffuse nel collagene e forma diverse strutture simili a quelli osservati in tumours1 umano. Adipociti vitali, le cellule epiteliali e fibroblasti all&#39;interno del nucleo originario erano evidenti sulla istologia. Maligna delle cellule epiteliali con morfologia squamoidi sono state dimostrate in invadendo il collagene circostante. Pleomorfismo nucleare era evidente all&#39;interno di queste cellule, insieme a figure mitotiche e corpi apoptotici. Abbiamo impiegato Tomografia Ottica di proiezione (OPT), una tecnologia di imaging 3D, al fine di quantificare l&#39;entità della diffusione del tumore nella cultura. Abbiamo usato OPT per misurare il volume della cultura di massa del tumore, un parametro di routine misurati durante la terapia neo-adiuvante dei pazienti con cancro al seno per valutare la risposta alla terapia farmacologica. Qui, vi presentiamo l&#39;opportunità di tumori al seno cultura umana, senza sotto-tipo di polarizzazione e quantificare la diffusione di questi ex-vivo. Questo metodo potrebbe essere utilizzato in futuro per quantificare la sensibilità ai farmaci nel tumore originale. Questo può fornire un modello più predittivo di linee cellulari attualmente in uso.

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

Abbiamo sviluppato una base di collagene<em&gt; In vitro</emTest&gt; che promuove la proliferazione e l&#39;invasione da campioni di tutti i sottotipi di tumore al seno. Tomografia ottica di proiezione, una tecnica di microscopia a tre dimensioni è stato utilizzato per visualizzare e quantificare l&#39;espansione del tumore. Il test può essere utilizzato per quantificare risposta ai farmaci dei singoli campioni tumorali.

A lungo termine Cultura di campioni umani cancro al seno e la loro analisi utilizzando la tomografia ottica di proiezione

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 Views.  University of Edinburgh. Abbiamo sviluppato una base di collagene In vitro che promuove la proliferazione e l'invasione da campioni di tutti i sottotipi di tumore al seno. Tomografia ottica di proiezione, una tecnica di microscopia a tre dimensioni è stato utilizzato per visualizzare e quantificare l'espansione del tumore. Il test può essere utilizzato per quantificare risposta ai farmaci dei singoli campioni tumorali.

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

A lungo termine Imaging cellule di mammifero utilizzando Wide-Field Microscopia

Ricerca

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.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

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.

La normalizzazione nata per fluorescenza Tomografia Ottica proiezione per l&#39;imaging tutto il cuore

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 la coltura di cellule staminali epiteliali da pelle umana

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 Cultura Vivo di cellule primarie umane tube di Falloppio epiteliale

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 lungo termine del test di tossicità letale con il crostaceo 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.

Arricchimento di serie staminali spermatogoni e cellule progenitrici (SSC) in Cultura per la derivazione di lungo termine Lines topo adulto 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.

Rilevamento somatici genetiche Alterazioni in campioni tumorali da Exon Capture e Sequencing 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.

Multistrato di montaggio per lungo termine foglio leggero Microscopia di 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, trasfezione e cultura a lungo termine di cardiomiociti per topi e ratti adulti

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