Lorna Renshaw (Edinburgh Meme Birimi), klinik örneklerden elde onu değerli yardım için teşekkür ederim. Tümör malzeme kullanımı Lothian Araştırma Etik Komitesi (08/S1101/41) tarafından onaylanmış ve Deneysel Kanser Tıp Merkezi programı (Edinburgh) himayesi altında elde edilmiştir. Bu çalışma, İskoç Finansman Konseyi tarafından desteklenmektedir. ADL Miss Margaret Butters Reekie Güven klinik arkadaşı.

1. Rat Tails Kollajen ayıklanıyor <ol><li> % 70 etanol içinde dondurulmuş taze sıçan kuyrukları yerleştirin. </li><li> Bir neşter açığa tendonlar ile örten cilt Deglove. </li><li> Tendon kuyruk ve sterilize etmek için% 70 etanol yer uzak soyun. </li><li> Toplanan tendon ve asetik asit (250ml 0,5 M asetik asit 1g tendon) transfer tartılır. 4 48hr karıştırın ° C. </li><li> 30 dakika 10.000 g / tendon asetik asit karışımı Santrifüj ve pelet atın. </li><li> Süpernatantı eşit miktarda% 10 (w / v) NaCl ekleyin ve karışımı 4 gecede bekletilmesi ° C. </li><li> G. 10.000 ilave bir 30 dakika için kollajen zengin, çözünmez alt katman ve centrifugate toplayın </li><li> Süspanse edin 4 0.25m asetik asit kollajen zengin malzeme ° C ve 4 ° C&#39;de 3 gün boyunca, günde iki kez diyaliz tampon değişen 1:1000 asetik asit karşı diyaliz. </li><li> Ayrıca 4 santrifüj (20.000 g 2saat için) ve mağaza kollajen çözümü sterilize ° C. </li><li> 1mg/ml bir stok konsantrasyonu steril 1:1000 asetik asit ilavesi ile gerektiği gibi kolajen sulandırınız. </li></ol> 2. 3D Testi Oluşturma 3D testi bir hücre hattı assay dayalı, daha önce 2 yayınladı. <ol><li> Çoklu çekirdek biyopsisi, invaziv meme kanseri için küratif cerrahi rezeksiyon zaman olan hastalarda rıza hasat edilir. </li><li> Göz, 1mm 3 parça halinde bir neşter kullanılarak çekirdek bölün. Düzeltin ve makroskopik farklı yağ atın. </li><li> MEGM Komple medya parçaları bırakın. Bu durumda doku kadar 1hr saklanabilir. </li><li> Buz 1mg/ml sıçan kuyruğu kollajen kollajen jel, mix oluşturmak için, 1:1000 filtreli asetik asit, 10x DMEM/F12 3:5:1:1 bir oran ve 0.22M NaOH 0.3 nihai kollajen konsantrasyonunu oluşturmak için mg / ml. </li><li> 37 ° 1.2ml, kollajen karışımı bir inkübatör içine 24 plaka yeri ve bireysel kuyulara enjekte ° C&#39;de 10 dakika süreyle jelleşme başlatmak için. </li><li> 10dk sonra kuvöz, 24 plaka kaldırın. Jelleşme kollajen tümör parçaları yerleştirin ve yavaşça 10μl bir pipet kullanarak jel merkezine onları itmek. 24 plaka 1hr inkübatör dönün. </li><li> ER + tümörleri için, 3.2x10 -10 M. bir konsantrasyon ile β-estradiol MEGM Komple medya ek Jel deneyleri 1hr jel sonra, her bir kuyunun içine dikkatlice takviye medya 1.2ml tanıtmak. Tahlil içinde son β-östradiol konsantrasyonu 3 meme dokusunun fizyolojik östrojen düzeylerini özetlemek 1.6x10 -10 M equilibriate. </li><li> Böylece medyanın her jel içinde serbestçe yüzen dairesel sağlayan her jel bırakmadan, her bir kuyunun iç kenarı etrafında bir 10μl pipet, çalıştırın. Kuvöz, 24 plaka dönün. </li><li> Exchange medya deney sona kadar haftada iki kez desteklenmelidir. Medya çıkarılması ve formalin fiksatif ekleyerek deney sonlandırın. </li></ol> 3. Optik Projeksiyon Tomografi için hazırlık Testi Antikor Boyama Biz tüm numune boyama 4 için daha önce yayınlanmış bir yöntem benimsedik . <ol><li> Formalin sabit örnekleri PBS içinde 30 dakika yıkayın. :% 0.05 Tween 20 (% 0.05 PBST) içeren PBS her aşamasında% 33,% 66 ve% 100 ≥ 15dk seyreltilmiş metanol kademeli jeller dehydrate. </li><li> DMSO::% 30 H 2 O otofloresans gidermek için oda sıcaklığında 24 saat için 2:01:03 &#39;lik bir oran (% 15 v / v H 2 O 2) 2 taze hazırlanmış MeOH doku inkübe edin. </li><li> Örnekleri metanol içerisinde 30 dakika için iki kez yıkayın. Numuneler -80 ° C oda sıcaklığında (RT) en azından geri 1hr her zaman ve doku derin kısımlarında bu antijenleri sağlamak için beş kez işlenir erişilebilir dondurun. </li><li> Seyreltici (her adımda% 33,% 66 ve 15 dakika için% 100) olarak metanol ile geri% 0.05 PBST doku rehidrate. </li><li> 24 saat için çözüm engelleme (% 0,05 PBST serum% 10) doku engelleyin. </li><li> 48hr için% 5 DMSO içeren engelleme çözüm 1.5ml içinde seyreltilmiş 1:500 konsantrasyonda anti-sitokeratin tavşan kullanarak, epitel içeriğini belirlemek için doku primer antikor ile inkübe edin. </li><li> 30 dakika her biri için% 0.05 PBST örnekleri dört kez yıkayın. </li><li> 48hr için% 5 DMSO içeren 1.5ml engelleme çözüm seyreltilmiş ikincil floresan antikor (1:100 keçi anti-tavşan Alexa555), doku inkübe edin. </li><li> Örnekleri 30 dakika her biri için% 0.05 PBST dört kez tekrar yıkayın. </li></ol> 4. Optik Projeksiyon Tomografi Optik projeksiyon tomografi, 15 milimetre 5 kalınlığında floresan biyolojik örneklerin yüksek çözünürlüklü 3 boyutlu görüntüler üretmek için önceden geliştirilmiş bir mikroskopi tekniği. <ol><li> Mount ssaklanmaktadır% 1 düşük erime noktası agaroz jel ve 24 saat için% 100 metanol kurutmak. Babb çözümü (1:2 Benzil alkol, Benzil benzoat) 48hr için örnek temizleyin ve OPT 3001M Tarayıcı (Bioptonics) kullanarak tarama. </li><li> Hem hedef ve otofloresans Cy3 filtre seti (uyarıcı 545nm/30 nm, yayıcı 610 / 75 nm) ve sırasıyla GFP1 filtre seti (uyarıcı 425nm/40 nm, yayıcı LP475 nm) kullanarak yakalanır. </li></ol> 5. İmar ve 3D kantifikasyon <ol><li> OPT tarafından satın açısal projeksiyonlar kesit dilimleri işgali tahlil aracılığıyla bir dizi oluşturmak için NRecon yazılımı (SkyScan) kullanarak yeniden olabilir. Bu yazılımı ücretsiz olarak indirmek için kullanılabilir. <a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> . </li><li> Volocity yazılım (Perkin Elmer) kullanarak yeniden inşa verileri analiz edin. Gösteri Ücretsiz yazılımı indirmek için kullanılabilir. <a href="http://www.cellularimaging.com/products/Volocity/demo/">http://www.cellularimaging.com/products/Volocity/demo/</a> . </li><li> Sitokeratin boyanarak epitel hacmi ölçmek için, hedef kanal eşiğe girişimi azaltmak için en az voksel yoğunluğu eşik ayarlayın. Biz mevcut parametrelerin ölçümü uygulaması listesini kullanarak 9000 au ampirik bu eşiğin tanımlanan orijinal çekirdek (autofluorescent kanal demarcated) süreklilik epitel hacmi sayısal Bu bize farklı arasındaki epitel akıbet hacimleri ölçmek için izin tümör örneği. </li></ol> 6. Temsilcisi Sonuçlar: Testin geliştirilmesi sırasında, toplam 52 meme kanseri biyopsi örneklerinde histopatoloji geniş bir yelpazesi ile kullanılmaktadır. Bunlardan &lt;% 10 (5 / 52) en az bir canlı işgali tahlil sağlamak için başarısız oldu. Testi yetmezliği, bakteriyel enfeksiyon ya da üstü ya da yetersiz tümör biyopsisinin malzeme nedeniyle oldu. Bu nedenle testin uygulanması, meme kanserinin belirli bir alt tipi ile sınırlı değildir. Tipik testleri (Şekil 1), 20 gün sonra kolajen kültür sabit ve sonradan sitokeratin boyandı. Volocity yazılım orijinal çekirdek ve müstakil hücreleri istila grupları ile sürekli epitel toplu hem de tanımak ve her birimin ayrı ayrı ölçmek için yapabiliyor. <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="Şekil 1" /> Fiksasyon ve miktar aşağıdaki Şekil 1 Invasion testleri. Birden fazla meme kanseri alt tiplerinin elde edilen numunelerin tahlil destekler: a) ER + b) HER2 + ve 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 ve JF MAM Teknoloji, Bioptonics OPT tarayıcılar ticarileştirmek tarafından istihdam edilmektedir. Diğer yazarlar, herhangi bir çıkar çatışması beyan ederim.

Kanser hücre hatları davranışlarını incelemek için kolajen bazlı testlerin artık yaygın olarak 6,7,8 kullanılır . Bununla birlikte, bu testlerin, tümör ve çevresinin tam karmaşıklığı yansıtılmaz. Bu çalışmada, insan meme kanseri malzemeleri, benzer bir biçimde, ex vivo kullanılabilir . Ayrıca, OPT, meme kanseri 3D genişleme karakterize etmek için yararlı bir araçtır. Biz bu tekniğin ana sınırlama tümör malzemeleri (tek bir biyopsi 4-6 deneyleri için yeterli) olduğu bulundu. Başka bir sınırlayıcı faktör yapısal işgali, sadece testlerin yaklaşık% 40 oluşur. Bunu aşmak için, düzenli olarak görünür büyüme ile 7 gün etrafında istenen herhangi bir tedavi tüm analizler eklemek. OPT kullanımı da toplu değişiklik, ex vivo, ilaç tedavisinden kaynaklanan algılamak için gelecekte uzatılabilir. Bu da daha bireyselleştirilmiş kanser tedavisi, hayvan modellerinde bağımsız kolaylaştırabilir. Şu anda ER + tümörler tamoksifen ex vivo yanıt keşfediyoruz.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog No. </td></tr><tr><td> Komple 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> ß-östradiol </td><td> Sigma-Aldrich </td><td> E8875-250mg </td></tr><tr><td> Tavşan, anti-sitokeratin </td><td> Dako </td><td> Z0622 </td></tr><tr><td> Keçi anti-tavşan Alexa555 </td><td> Invitrogen </td><td> A21422 </td></tr></tbody></table> Tüm diğer standart reaktifler Sigma-Aldrich elde edildi.

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

Meme kanseri, Batı dünyasının önde gelen ölüm nedenidir. Bu, ilk önce meme kanserinin yayılmasını yerel ve daha sonra distalde, hasta prognozu önemli bir faktör olduğunu kurulmuştur. Deneysel meme kanseri sistemleri, genellikle birincil tümörler veya plevral efüzyon türetilen hücre hatları güveniyor. Iki büyük engel bu araştırma engel: (i) bazı bilinen meme kanseri (özellikle kötü prognoz luminal B tümörler) alt türleri mevcut hat koleksiyonları içinde temsil edilmemektedir, (ii), tümör mikroçevresinin etkisi genellikle dikkate alınmaz. Biz tüm alt türleri kültür primer meme kanseri örneklerinde bir teknik göstermektedir. Bu tümör küçük parçalar Ben yastıklar, yumuşak sıçan kollajen gömülü olduğu üç boyutlu (3D) kültür sistemi kullanılarak elde edilir. 2-3 hafta içinde, tümör hücreleri, kollajen yayılmış ve insan tumours1 görülenlere benzer çeşitli yapılar oluşturur. Canlı adipositler, orijinal çekirdek içinde epitel hücreleri ve fibroblastlar histoloji belirgindi. Squamoid morfolojisi malign epitel hücreleri çevreleyen kolajen içine işgalci gösterildi. Nükleer pleomorfizm, mitotik figürler ve apoptotik organları ile birlikte, bu hücrelerin içinde açıkça görülmüştür. Biz kültür tümörün yayılma derecesi ölçmek için Optik Projeksiyon Tomografi (OPT), 3D görüntüleme teknolojisine, istihdam var. Biz, tümör kültür toplu hacmini ölçmek için OPT, bir parametre, ilaç tedavisine yanıtı değerlendirmek için rutin meme kanseri hastalarının, neo-adjuvan tedavi sırasında ölçülen. Burada alt türü önyargısız kültür insan göğüs tümörleri bir fırsat ve bu ex vivo yayılmasını ölçmek. Bu yöntem gelecekte orijinal tümör ilaç duyarlılığını ölçmek için kullanılan olabilir. Bu, şu anda kullanılan hücre hatları daha prediktif model sağlayabilir.

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

Biz bir kolajen bazlı geliştirdik<em&gt; In vitro</em&gt; Tüm meme kanseri alt tiplerinin örnekleri çoğalması ve işgali teşvik tahlil. Optik Projektör Tomografi, üç boyutlu mikroskopi tekniği tümör genişlemesi görselleştirmek ve ölçmek için kullanılmıştır. Bu testte, bireysel tümör örneklerinin ilaç tepkisini ölçmek için kullanılıyor olabilir.

Optik Projeksiyon Tomografi İnsan Meme Kanseri Örnekler ve Analiz Uzun vadeli Kültür

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

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Biology

14.6K Görüntüleme.  University of Edinburgh. Biz bir kolajen bazlı geliştirdik In vitro Tüm meme kanseri alt tiplerinin örnekleri çoğalması ve işgali teşvik tahlil. Optik Projektör Tomografi, üç boyutlu mikroskopi tekniği tümör genişlemesi görselleştirmek ve ölçmek için kullanılmıştır. Bu testte, bireysel tümör örneklerinin ilaç tepkisini ölçmek için kullanılıyor olabilir.

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

Geniş Alan Mikroskop kullanarak Uzun vadeli Görüntüleme Memeli Hücreleri

Biyoloji

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.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

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.

Tüm Kalp Görüntüleme Floresan Optik Projeksiyon Tomografi tarihi Normalizasyon

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.

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

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 Kültür İlköğretim İnsan Fallop Tüp Epitel Hücreleri

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.

Kabuklu uzun vadeli Lethal Toksisite Testi 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.

Uzun vadeli Yetişkin Fare SSC Hatları türetilmesi için Kültür Spermatogonial Kök ve progenitör hücrelerin Seri Zenginleştirme (DGM)

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.

Ekson yakalama ve Devasa Paralel Dizilimine tarafından Tümör Örneklerinin somatik genetik değişiklikler tespit

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.

Çok katmanlı Zebra balığı uzun vadeli Işık Sayfalık Mikroskopi için Montaj

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.

İzolasyon, Transfeksiyon ve Yetişkin Fare ve Sıçan Kardiyomiyositlerinin Uzun Süreli Kültürü

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