Name: an ex vivo culture system to study thyroid development
Uploaded: 2014-06-06T13:45:00
Description: Watch this Scientific Journal Video about An Ex vivo Culture System to Study Thyroid Development at JoVE.com

The work was supported by grants from the Universit&#233; catholique de Louvain (Action de recherch&#233; concert&#233;es) and the Fund for Scientific Medical Research (F.R.S.-FNRS, Belgium). A.-S.D. is a doctoral fellow from T&#233;l&#233;vie, A.-C.H. held a fellowship from the Fonds pour la formation &#224; la Recherche dans l'Industrie et dans l'Agriculture (Belgium), M.V. is supported by the de Duve Institute and C.E.P. is a senior research associate of the F.R.S.-FNRS (Belgium).

I topi sono stati allevati e trattati secondo i principi della cura degli animali da laboratorio della commissione per il Benessere degli Animali University. Tutte le procedure e protocolli sono stati approvati dal comitato. 1. Rivestimento di Microscopia plastica Cultura Chambers NOTA: Eseguire tutte le seguenti operazioni in condizioni di sterilità in una cappa a flusso laminare. I due tipi di rivestimento sono funzionalmente equivalenti. <ol><li> Cappotto d...

<ol>
	<li>Colin, I. M., Denef, J. -. F., Lengel&#233;, B., Many, M. -. C., G&#233;rard, A. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+the+cell+biology+of+thyroid+angiofollicular+units.">Recent insights into the cell biology of thyroid angiofollicular units.</a> Endocr. Rev. 34 (2), 209-238 (2013).</li><li>Fagman, H., Andersson, L., Nilsson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developing+mouse+thyroid+embryonic+vessel+contacts+and+parenchymal+growth+pattern+during+specification+budding,+migration,+and+lobulation.">The developing mouse thyroid embryonic vessel contacts and parenchymal growth pattern during specification budding, migration, and lobulation.</a> Dev. 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Rev. 25 (5), 722-746 (2004).</li><li>De Felice, M., Di Lauro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+extrinsic+factors+in+thyroid+gland+development:+an+update.">Intrinsic and extrinsic factors in thyroid gland development: an update.</a> Endocrinology. 152 (8), 2948-2956 (2011).</li><li>Hick, A. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+epithelial+paracrine+interactions+during+thyroid+development+govern+follicular+organization+and+C-cells+differentiation.">Reciprocal epithelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation.</a> Dev. Biol. 381 (1), 227-240 (2013).</li><li>Toda, S., Koike, N., Sugihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+integration+of+thyrocytes+and+thyroid+folliculogenesis:+a+perspective+for+thyroid+tissue+regeneration+and+engineering.">Cellular integration of thyrocytes and thyroid folliculogenesis: a perspective for thyroid tissue regeneration and engineering.</a> Endocr. J. 48, 407-425 (2001).</li><li>Toda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+models+for+studying+thyroid+biology+and+disorders.">Culture models for studying thyroid biology and disorders.</a> ISRN Endocrinol. , (2011).</li><li>Eggo, M. C., Quiney, V. M., Campbell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+factors+regulating+growth+and+function+of+human+thyroid+cells+in+vitro+and+in+vivo.">Local factors regulating growth and function of human thyroid cells in vitro and in vivo.</a> 213, 47-58 (2003).</li><li>Antonica, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+thyroid+from+embryonic+stem+cells.">Generation of functional thyroid from embryonic stem cells.</a> Nature. 491, 66-71 (2012).</li><li>van Eyll, J. M., Pierreux, C. E., Lemaigre, F. P., Rousseau, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shh-dependent+differentiation+of+intestinal+tissue+from+embryonic+pancreas+by+activin+A.">Shh-dependent differentiation of intestinal tissue from embryonic pancreas by activin A.</a> J. Cell Sci. 117, 2077-2086 (2004).</li><li>Hick, A. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+primitive+duct+formation+in+the+pancreas+and+submandibular+glands:+a+role+for+SDF-1.">Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1.</a> BMC Dev. Biol. 9. , (2009).</li><li>Pierreux, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial:Endothelial+cross-talk+regulates+exocrine+differentiation+in+developing+pancreas.">Epithelial:Endothelial cross-talk regulates exocrine differentiation in developing pancreas.</a> Dev. Biol. 347, 216-227 (2010).</li><li>Puri, S., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+embryonic+pancreas+development+using+real-time+imaging.">Dynamics of embryonic pancreas development using real-time imaging.</a> Dev. Biol. 306, 82-93 (2007).</li><li>Petzold, K. M., Spagnoli, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+ex-vivo+culturing+of+embryonic+pancreas.">A system for ex-vivo culturing of embryonic pancreas.</a> J. Vis. Exp. , (2012).</li><li>Watanabe, T., Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+ureteric+bud+branching+morphogenesis+in+vitro.">Real-time analysis of ureteric bud branching morphogenesis in vitro.</a> Dev. Biol. 271, 98-108 (2004).</li></ol>

Questo documento descrive un metodo per la dissezione e la coltura espianti tiroidei (E12.5) o lobi (E14.5), al fine di studiare e comprendere meglio le complesse vicende che portano alla formazione della tiroide. Grazie alla duplice origine del anlagen tiroide (anlage linea mediana e due corpi laterali ultimobranchial), e le loro piccole dimensioni, un frammento di tessuto E12.5 rostrale localizzato alle arterie arco faringeo, e contenente la trachea, ma non l&#39;esofago è microdissezione. Come illustrato, entro un p...

La tiroide è una raccolta di sfere epiteliali indipendenti, chiamati follicoli, circondato da una fitta rete di capillari endoteliali. Questa organizzazione permette la funzione della tiroide: i capillari endoteliali forniscono tireociti con iodio, necessari per T3 e T4 sintesi degli ormoni, e distribuire questi ultimi a tutto il corpo. Sparsi tra i follicoli e dei capillari, C-cellule producono l&#39;ormone calcitonina hypocalcemic 1. Anche se l&#39;architettura della tiroide adulti e le funzioni sono ben noti, i meccanismi cellulari e molecolari coinvolti nella tiroide sviluppo embrionale (formazione del follicolo e differenziazione) sono lungi dall&#39;essere compreso. Durante l&#39;embriogenesi, tireociti progenitore nasce come un ispessimento (anlage linea mediana) della parete ventrale del endoderma foregut al giorno embrionale (e) 8,5 nell&#39;embrione topo, mentre cellule C progenitori origine a E11.5 come sporgenze a forma di gocciolina ( bo ultimobranchialmuore), della quarta tasche faringee 2-6. La gemma linea mediana stacca quindi dal endoderma, si espande bilateralmente a fondere a E13.5 con gli organi ultimobranchial su ogni lato della trachea. Infine, tireociti organizzano in follicoli e C-cellule si differenziano. Le conoscenze attuali sulla formazione tiroide proviene principalmente da analisi istologica dei tessuti fissi, ma gli eventi morfogenetici coinvolti nella formazione tiroide sono altamente dinamico e coinvolgere comunicazioni e le interazioni tra diversi tipi di cellule e con la matrice extracellulare. Lavoro recente ha dimostrato che progenitori tireocita producono alti livelli di VEGF per reclutare cellule endoteliali alla tiroide sviluppo, e, a sua volta, reclutati cellule endoteliali promuovere la formazione del follicolo e cellule C differenziazione 7. La maggior parte degli studi ex vivo sulla tiroide sono stati effettuati su cellule isolate adulte tiroide-derivato, coltivate in 2D coltura tissutale di plastica drocchie o in matrici 3D. L&#39;utilizzo di questi tipi di culture, le cellule follicolari differenziate o rimanere polarizzata e organizzate come i follicoli o riacquisire un organismo 3D 8-10. Tuttavia, queste cellule epiteliali puri, espiantati dal loro ambiente fisiologico, e coltivate in 2D, ignorano interazioni con matrice extracellulare, citochine, fattori di crescita e con altri tipi cellulari, quali le cellule endoteliali o nervi che normalmente incontrano in vivo. Un bel studio recentemente descritto un protocollo di differenziazione delle cellule staminali in follicoli tiroidei con un passo di coltura in 3D matrigel 11. Tuttavia, queste culture 3D mancano contatto con altri tipi di cellule. Sulla base delle esperienze precedenti su pancreas e ghiandole salivari cultura d&#39;organo 12-14, un metodo per la dissezione del mouse embrionali anlagen tiroide e coltivando gli espianti sui filtri semiporoso o su scivoli di plastica microscopia stato sviluppato. Quandolavorare con embrioni E12.5, la doppia origine del abbozzi tiroide (il anlage linea mediana e due corpi laterali ultimobranchial) ha imposto la microdissezione di un grande frammento di tessuto. Questo conteneva la trachea, ma non l&#39;esofago, ed esteso dalle arterie arco faringeo fino a soffiatura aritenoidea. Quando coltivate su filtri, la anlage linea mediana si estende lateralmente su ciascun lato della trachea, dove si fondono con gli organi ultimobranchial per formare i due lobi tiroidei, ancora collegati da uno stretto istmo. Nella cultura, cellule epiteliali proliferano, organizzano in follicoli e si differenziano in tireociti e cellule C, a seconda della loro origine. Le cellule endoteliali contenute nel tessuto microdissezione anche proliferano e invadono i lobi tiroidei associare finalmente stretto contatto con la struttura follicolare epiteliale, indipendentemente flusso sanguigno o fattori circolanti. Come sviluppo degli espianti riassume fedelmente in vivo svilupparezione, questo sistema di coltura è ottimale per studiare morfogenetica e gli eventi che si verificano durante lo sviluppo differenziazione tiroidea. Tessuti tiroidei possono essere ottenute da wild type, knockout o embrioni transgenici fluorescenti, e il sistema cultura è suscettibile di perdita e di guadagno-di-funzione esperimenti. Infine, time-lapse imaging di espianti tiroidei fluorescente su piatti di plastica microscopia potrebbe essere sfruttata per studiare meglio la cinetica e la continuità dei movimenti morfogenetici che si verificano in vivo. Time-lapse imaging è già stata utilizzata per studiare branching morfogenesi del pancreas 15,16 o la gemma ureterale 17.

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			<td height="21" style="height:21px;width:327px;">&#181;-slide 8 well, ibiTreat, tissue culture treated, sterile</td>
			<td style="width:85px;">proxylab</td>
			<td style="width:119px;">80826</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type I, rat tail</td>
			<td style="width:85px;">Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin from human plasma</td>
			<td style="width:85px;">Invitrogen</td>
			<td># 33010-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">M199</td>
			<td style="width:85px;">Invitrogen</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">HBSS</td>
			<td style="width:85px;">Invitrogen</td>
			<td>14025-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Tungsten wire</td>
			<td style="width:85px;">Goodfellow</td>
			<td style="width:119px;">LS237450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">culture plate insert (12 mm diameter)</td>
			<td style="width:85px;">Millipore</td>
			<td>PICM01250</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">GlycoBlue</td>
			<td style="width:85px;">Invitrogen</td>
			<td style="width:119px;">AM9516</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">E-cadherin</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">610182</td>
			<td>1/1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Ezrin</td>
			<td style="width:85px;">Thermo Scientific</td>
			<td style="width:119px;">MS-661-P1</td>
			<td>1/400</td>
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			<td height="42" style="height:42px;width:327px;">PECAM</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">550274</td>
			<td>1/100</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Hoechst</td>
			<td style="width:85px;">Sigma</td>
			<td style="width:119px;">B2261</td>
			<td></td>
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</table>

an ex vivo culture system to study thyroid development

La tiroide è una ghiandola endocrina bilobated localizzata alla base del collo, producendo gli ormoni tiroidei T3, T4, e calcitonina. T3 e T4 sono prodotti da tireociti differenziati, organizzati in sfere chiusi chiamati follicoli, mentre calcitonina è sintetizzato dalle cellule C, intervallati tra i follicoli e una fitta rete di capillari sanguigni. Sebbene architettura tiroide adulto e funzioni sono state ampiamente descritte e studiato, la formazione delle unità &quot;angio-follicolari&quot;, la distribuzione delle cellule C nel parenchima e le comunicazioni paracrini tra cellule epiteliali ed endoteliali è lungi dall&#39;essere compresa. Questo metodo descrive le fasi sequenziali di topo tiroide embrionale anlagen dissezione e la sua cultura sui filtri semiporoso o su scivoli di plastica microscopia. Entro un periodo di quattro giorni, questo sistema di coltura riassume fedelmente nello sviluppo della tiroide vivo. Infatti, (i) bilobation dell&#39;organo verifica (per espianti E12.5), (ii) tireociti precursori organizzano in follicoli e polarizzano, (iii) tireociti e C-cellule si differenziano, e (iv) le cellule endoteliali presenti nel tessuto microdissezione proliferano, migrano nella lobi tiroidei, e strettamente associano con le cellule epiteliali, come fanno in vivo. Tessuti tiroidei possono essere ottenute da wild type, knockout o embrioni transgenici fluorescenti. Inoltre, espianti coltura può essere manipolato per aggiunta di inibitori, bloccando anticorpi, fattori di crescita, o anche cellule o mezzo condizionato. Ex vivo sviluppo può essere analizzato in tempo reale, o in qualsiasi momento della coltura mediante immunoistochimica e RT-qPCR. In conclusione, la cultura espianto tiroide combinato con a valle intera-mount o su sezioni di imaging e di espressione genica fornisce un potente sistema per manipolare e studiare morfogenetiche e differenziazione eventi della tiroide organogenesis.

Anlages tiroide (linea mediana e UB, insieme con i tessuti circostanti) e lobi tiroidei sono sezionati da embrioni di topo a E12.5 e e13.5/e14.5, rispettivamente (Figura 1). Dopo un giorno di coltura sul filtro, la anlage linea mediana è visibile (Figura 2A) come un tessuto allungata si estende sulla parte superiore della trachea. Esso forma progressivamente due lobi su ciascun lato della trachea. Se lobi tiroidei isolati (E13.5 o E14.5) sono coltivate, si espandono, subiscono morfogen...

Watch this Scientific Journal Video about An Ex vivo Culture System to Study Thyroid Development at JoVE.com

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Un<em&gt; Ex vivo</em&gt; Cultura sistema per studiare lo sviluppo della tiroide

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

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An Ex vivo Culture System to Study Thyroid Development

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Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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Biologia

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Un Ex-ovo Sistema di embrioni di pollo Cultura Adatto per applicazioni di imaging e Microchirurgia

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

Chiuso Sistema Cultura protocollo cellulare con imbarcazioni HyperStack con la tecnologia dei materiali gas permeabili

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embrionali di topo di sviluppo in un embrione di tutta la cultura di sistema privo di siero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Mouse fetale Whole Intestino Sistema Cultura per<em&gt; Ex Vivo</em&gt; La manipolazione delle vie di segnalazione e tridimensionale Imaging in diretta di Sviluppo del villo

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Sviluppo di un sistema di inserimento co-coltura di due tipi cellulari in assenza del cellula-cellula di contatto

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Istituzione di un pH extracellulare acido sistema cultura

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...