Wir danken Lorna Renshaw (Edinburgh Breast Unit) für ihre unschätzbare Hilfe bei der Beschaffung von klinischen Proben. Verwendung von Tumormaterial wurde von der Lothian Research Ethics Committee (08/S1101/41) zugelassen und erhältlich unter der Schirmherrschaft des Experimental Cancer Medicine Centre Programm (Edinburgh). Diese Arbeit wird von der Scottish Funding Council unterstützt. ADL ist eine Miss Margaret Butters Reekie Vertrauen klinischen Kollegen.

1. Extrahieren von Collagen aus Rattenschwänzen <ol><li> Legen Sie gefrorenes Frischplasma Rattenschwänzen in 70% Ethanol. </li><li> Deglove die darüber liegende Haut mit einem Skalpell auszusetzen Sehnen. </li><li> Streifen Sehnen weg vom Schwanz und in 70% Ethanol zu sterilisieren. </li><li> Wiegen Sie die gesammelten Sehnen und Transfer zu Essigsäure (1g Sehne 250ml 0,5 M Essigsäure). Mix für 48hr bei 4 ° C. </li><li> Centrifuge die Sehne / Essigsäure-Mix bei 10.000 g für 30min und entsorgen Sie die Pellets. </li><li> Fügen Sie dem gleichen Volumen 10% (w / v) NaCl, um den Überstand und lassen Sie die Mischung über Nacht stehen bei 4 ° C. </li><li> Sammeln Sie die Kollagen-reich, unlöslich Bodenschicht und Zentrifugat für weitere 30 Minuten bei 10.000 g. </li><li> Resuspendieren Kollagen-reiches Material in 0,25 M Essigsäure bei 4 ° C und Dialyse gegen 1:1000 Essigsäure bei 4 ° C für 3 Tage, Wechsel der Dialyse-Puffer zweimal täglich. </li><li> Weitere sterilisieren Kollagenlösung durch Zentrifugation (20.000 g für 2 Std.) und lagern bei 4 ° C. </li><li> Verdünnen Kollagen mit der Zugabe von sterilem 1:1000 Essigsäure auf einen Bestand Konzentration von 1mg/ml erforderlich. </li></ol> 2. 3D-Assay Creation Die 3D-Test auf eine Zelllinie Assay basiert, veröffentlicht bisher 2. <ol><li> Mehrere Stanzbiopsien aus zustimmenden Patienten zum Zeitpunkt der kurativen Resektion für invasiven Brustkrebs geerntet. </li><li> Mit Auge, teilen sich die Kerne mit einem Skalpell in 1mm 3 Fragmente. Trim und entsorgen makroskopisch deutlich Fett. </li><li> Tauchen Sie die Fragmente in MEGM Komplett Media. Das Gewebe kann in diesem Zustand gelagert werden bis zu 1 Stunde. </li><li> Zum Erstellen der Kollagen-Gel, auf Eis 1mg/ml Rattenschwanz Collagen, bis 1:1000 gefiltert Essigsäure, 10x DMEM/F12 und 0,22 M NaOH in einem Verhältnis von 3:5:1:1 erstellen eine abschließende Kollagen-Konzentration von 0,3 mg / ml. </li><li> Inject 1.2ml von Kollagen-Gemisch in die einzelnen Wells einer 24-Well-Platte aufnehmen und in einem Brutschrank bei 37 ° C für 10 Minuten, um ein Gelieren zu initiieren. </li><li> Nach 10min, entfernen Sie den 24-Well-Platte aus dem Inkubator. Ort des Tumors Stücke auf die Gelier-Kollagen und schieben sie in die Mitte des Gels mit einem 10 &amp;mgr; l Pipettenspitze. Gibt die 24-Well-Platte in den Inkubator für 1 Stunde. </li><li> Für ER + Tumore, zu ergänzen MEGM komplette Medien mit β-Östradiol zu einer Konzentration von 3.2x10 -10 M. Nachdem das Gel Assays für 1 Stunde haben geliert, sorgfältig einführen 1.2ml der ergänzten Medien in jedes Well. Die endgültige β-Östradiol-Konzentration im Assay wird 1.6x10 -10 M equilibriate auf physiologische Östrogenspiegel im Brustgewebe 3 gefunden zu rekapitulieren. </li><li> Führen Sie einen 10 &amp;mgr; l Pipettenspitze um den inneren Rand jeder gut, die Freigabe jedes Gel Umfang, so dass jedes Gel frei schweben in den Medien. Gibt die 24-Well-Platte in den Inkubator. </li><li> Börse ergänzt Medien zweimal wöchentlich bis zur Beendigung des Experiments. Beenden Sie den Versuch durch Entfernen von Medien und das Hinzufügen einer Formalin Fixativ. </li></ol> 3. Antikörper-Färbung von Assay in Vorbereitung für Optical Projection Tomographie Wir haben eine zuvor veröffentlichte Methode zur gesamten Stichprobe Färbung 4 angenommen. <ol><li> Wash Formalin fixierten Proben für 30min in PBS. Entwässern der Gele schrittweise in Methanol in PBS mit 0,05% Tween 20 (0,05% PBST) verdünnt: 33%, 66% und 100% für ≥ 15min bei jedem Schritt. </li><li> Inkubieren des Gewebes in frisch zubereiteten MeOH: DMSO: 30% H 2 O 2 in einem Verhältnis von 02.01.03 (15% v / v H 2 O 2) bei Raumtemperatur für 24 Stunden auf die Autofluoreszenz zu stillen. </li><li> Wash-Proben zweimal für 30 Minuten in Methanol. Einfrieren der Proben bis -80 ° C fünf Mal für mindestens 1 Stunde jedes Mal und wieder auf Raumtemperatur (RT) auf, die Antigene in den tieferen Teilen des Gewebes gewährleistet werden zugänglich gemacht. </li><li> Rehydrieren das Gewebe wieder auf 0,05% PBST mit Methanol als Verdünnungsmittel (33%, 66% und 100% für 15 Minuten bei jedem Schritt). </li><li> Block des Gewebes in Blocking-Lösung (10% Serum in 0,05% PBST) für 24 Stunden. </li><li> Inkubieren des Gewebes mit primärer Antikörper gegen das epitheliale Inhalt zu identifizieren, mit Kaninchen-Anti-Cytokeratin in einer Konzentration von 1:500 in 1,5 ml der Sperrung Lösung mit 5% DMSO für 48hr verdünnt. </li><li> Wash Proben viermal mit 0,05% PBST 30 min je. </li><li> Inkubieren des Gewebes mit fluoreszierenden sekundären Antikörper (1:100 Ziege Anti-Kaninchen-Alexa555), in 1,5 ml Blocking-Lösung mit 5% DMSO für 48hr verdünnt. </li><li> Waschen Sie die Proben wieder mit 0,05% PBST viermal für jeweils 30 Minuten. </li></ol> 4. Optical Projection Tomographie Optische Projektions-Tomographie ist ein Mikroskopie-Technik bereits entwickelt, um hochauflösende 3D-Bilder von fluoreszierenden biologischen Proben mit einer Dicke von bis zu 15 Millimeter 5 zu erzeugen. <ol><li> Montieren Sie die stained Gel in 1% Agarose für niedrigen Schmelzpunkt und entwässern in 100% Methanol für 24 Stunden. Deaktivieren Sie die Probe in BABB Lösung (1:2 Benzylalkohol, Benzylbenzoat) für 48hr und scannen mit dem OPT 3001M Scanner (Bioptonics). </li><li> Sowohl Ziel-und Autofluoreszenz erfasst mit dem Cy3-Filtersatz (Erreger 545nm/30 nm, Emitter 610/75 nm) und GFP1 Filter-Set (Erreger 425nm/40 nm, Emitter LP475 nm) bzw.. </li></ol> 5. Wiederaufbau-und 3D-Quantifizierung <ol><li> Der Satz von eckigen Projektionen OPT erworbene rekonstruiert mit NRecon Software (SkyScan) zu einer Reihe von Querschnitt schneidet durch die Invasion Assay zu erzeugen. Diese Software ist für Sie kostenlos bei Download <a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> . </li><li> Analyse der rekonstruierten Daten mit Volocity Software (PerkinElmer). Kostenlose Demo-Software ist zum Download verfügbar unter <a href="http://www.cellularimaging.com/products/Volocity/demo/">http://www.cellularimaging.com/products/Volocity/demo/</a> . </li><li> Um epithelialen Volumen von Zytokeratin-Färbung zu quantifizieren, stellen ein Minimum Voxel Intensität Schwelle für Störungen durch Hintergrundfluoreszenz in der Ziel-Kanal zu reduzieren. Wir definierten diese Schwelle empirisch bei 9000 au Mit einer Liste der verfügbaren Parameter in der Messaufgabe, wir das Volumen des Epithels in Kontinuität mit dem ursprünglichen Kern (abgegrenzt im autofluoreszierenden Kanal) quantifiziert. Dies erlaubt uns zu einem Volumen von epithelialen Auswuchs zwischen verschiedenen quantifizieren Tumorproben. </li></ol> 6. Repräsentative Ergebnisse: Bei der Entwicklung des Tests, Biopsie insgesamt 52 Brustkrebs Proben mit einer breiten Palette von Histopathologie verwendet wurden. Von diesen &lt;10% (5 / 52) nicht mindestens eine tragfähige Invasion Assay liefern. Assay Misserfolg war entweder aufgrund bakterieller supra-Infektion oder unzureichende Tumor Biopsie-Material. Deshalb Anwendung des Tests ist nicht an einen bestimmten Subtyp von Brustkrebs beschränkt. Typische Assays (Abbildung 1) nach 20 Tagen in Kollagen Kultur fixiert und anschließend für Cytokeratin gefärbt. Volocity Software ist in der Lage, sowohl die epithelialen Masse kontinuierlich mit dem ursprünglichen Kern und freistehende Gruppen von eindringenden Zellen zu erkennen und zu quantifizieren, jedes Volume separat. <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Invasion Assays folgende Fixierung und Quantifizierung. Der Test unterstützt Proben aus verschiedenen Brustkrebs-Subtypen erhalten: a) ER + b) HER2 + und 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 und JF von MRC-Technologie, welche die Kommerzialisierung Bioptonics OPT-Scanner eingesetzt wird. Andere Autoren erklären, keinen Interessenkonflikt.

Collagen-basierte Assays, um das Verhalten von Krebszelllinien Studie sind jetzt weit 6,7,8 verwendet. Dennoch haben diese Tests nicht in vollem Umfang die Komplexität des Tumors und seiner Umgebung wider. In dieser Studie zeigen wir, dass menschliche Brustkrebszellen Materialien in ähnlicher Weise, ex vivo eingesetzt werden kann. Darüber hinaus ist OPT ein nützliches Werkzeug, um die 3D-Erweiterung von Brustkrebs zu charakterisieren. Wir fanden, dass die wichtigste Einschränkung dieser Technik die Verfügbarkeit von Tumor-Materialien (a Single-Core-Biopsie ist ausreichend für 4-6 Assays) ist. Ein weiterer limitierender Faktor ist, dass strukturelle Invasion tritt nur in etwa 40% des Assays. Um dies zu überwinden, wir regelmäßig add beliebige Behandlung um Tag 7, um alle Tests mit sichtbarem Wachstum. Der Einsatz von OPT könnte in der Zukunft erweitert werden, um auch erkennen, Bulk-Änderungen, ex vivo, die sich aus medikamentöser Behandlung. Dies wiederum könnte zu erleichtern stärker individualisierten Krebstherapie, unabhängig von Tiermodellen. Wir prüfen derzeit die Antwort der ER + Tumore auf Tamoxifen ex vivo.

<table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Katalog Nr. </td></tr><tr><td> MEGM Komplette </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-Östradiol </td><td> Sigma-Aldrich </td><td> E8875-250MG </td></tr><tr><td> Rabbit anti-Cytokeratin </td><td> Dako </td><td> Z0622 </td></tr><tr><td> Ziege Anti-Kaninchen-Alexa555 </td><td> Invitrogen </td><td> A21422 </td></tr></tbody></table> Alle anderen Standard-Reagenzien wurden von Sigma-Aldrich bezogen.

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

Brustkrebs ist eine der Hauptursachen für die Sterblichkeit in der westlichen Welt. Es ist bekannt, dass die Ausbreitung von Brustkrebs, zunächst lokal und dann distal, ist ein wichtiger Faktor in die Prognose der Patienten. Experimental-Systeme von Brustkrebs vertrauen auf Zell-Linien in der Regel von Primärtumoren oder Pleuraergüsse abgeleitet. Zwei große Hindernisse stehen dieser Forschung: (i) einige bekannte Sub-Typen von Brustkrebs (vor allem schlechte Prognose luminalen B Tumoren) nicht innerhalb der aktuellen Zeile Sammlungen vertreten; (ii) den Einfluss der Tumor-Mikroumgebung in der Regel nicht berücksichtigt. Wir demonstrieren eine Technik zur Kultur primärem Mammakarzinom Proben aller Sub-Typen. Dies wird durch die dreidimensionale (3D-) Kultur, in dem kleine Stücke des Tumors in weichen Ratte Kollagen I Kissen eingebettet sind erreicht. Innerhalb von 2-3 Wochen, die Tumorzellen in das Kollagen verteilt und bilden verschiedene Strukturen ähnlich denen in der menschlichen tumours1 beobachtet. Tragfähige Adipozyten wurden Epithelzellen und Fibroblasten in den ursprünglichen Kern der Histologie evident. Bösartige Epithelzellen mit squamoid Morphologie wurden gezeigt Invasion in das umgebende Kollagen. Kernpolymorphie war in diesen Zellen evident, zusammen mit Mitosen und apoptotische Körper. Wir haben Optical Projection Tomographie (OPT), ein 3D-Imaging-Technologie, eingesetzt, um das Ausmaß der Tumorausbreitung in Kultur zu quantifizieren. Wir haben OPT verwendet werden, um den Großteil Volumen des Tumors Kultur zu messen, einen Parameter routinemäßig während der neo-adjuvanten Behandlung von Brustkrebs-Patientinnen gemessen werden, um als Reaktion auf medikamentöse Therapie zu beurteilen. Hier präsentieren wir die Gelegenheit, Kultur menschlichen Brusttumoren ohne sub-type Bias und zu quantifizieren, die Ausbreitung dieser ex vivo. Diese Methode könnte in der Zukunft genutzt werden, um Drogen-Empfindlichkeit in ursprünglichen Tumor zu quantifizieren. Dies kann einen besseren prädiktiven Modells als derzeit verwendete Zelllinien.

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

Wir haben eine Kollagen-basierten entwickelt<em&gt; In-vitro-</em&gt; Assay, der Proliferation und Invasion fördert von Proben aller Brustkrebs-Subtypen. Optical Projection Tomographie, eine dreidimensionale Mikroskopie-Technik wurde verwendet, um zu visualisieren und zu quantifizieren Tumorausdehnung. Dieser Test kann verwendet werden, um Drogen-Reaktion der einzelnen Tumorproben zu quantifizieren.

Langfristige Culture of Human Breast Cancer Proben und deren Analyse mit Hilfe von optischen Projektion Tomography

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 Aufrufe.  University of Edinburgh. Wir haben eine Kollagen-basierten entwickelt In-vitro- Assay, der Proliferation und Invasion fördert von Proben aller Brustkrebs-Subtypen. Optical Projection Tomographie, eine dreidimensionale Mikroskopie-Technik wurde verwendet, um zu visualisieren und zu quantifizieren Tumorausdehnung. Dieser Test kann verwendet werden, um Drogen-Reaktion der einzelnen Tumorproben zu quantifizieren.

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Langfristige Imaging Säugetierzellen mit Wide-Field-Mikroskopie

Forschung

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.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

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.

Geboren Normalisierung für Fluoreszenz-Optical Projection Tomographie für ganzem Herzen Imaging

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.

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

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-Kultur von primären humanen Eileiter Epithelzellen

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.

Langfristige letale Toxizität Test mit dem Krustentier 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.

Serielle Anreicherung von Spermatogonien Stamm-und Progenitorzellen (SSC) in der Kultur für die Ableitung des langfristigen adulten Maus SSC Linien

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.

Erkennen Somatische Genetische Veränderungen in Tumorproben von Exon-Abscheidung und-Massively Parallel Sequencing

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.

Multilayer-Halterung für Langzeit-Licht-Mikroskopie Blatt der Zebrafisch

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, Transfektion und Langzeitkultur von Erwachsenenmaus und Rattenkardiomyozyten

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