Il lavoro è stato supportato da sovvenzioni della Manpei Suzuki Diabetic Foundation (YT), del Boston Children's Hospital OFD/BTREC/CTREC Faculty Career Development Grant, della Boston Children's Hospital Ophthalmology Foundation, del BCH Pilot Award, della BCH Manton Center Fellowship e della Little Giraffe Foundation (ZF), della German Research Foundation (DFG; a BC [CA1940/1-1]), NIH R24EY024868, EY017017, R01EY01717-13S1, EY030904-01, BCH IDDRC (1U54HD090255), Massachusetts Lions Eye Foundation (LEHS).

Tutti gli esperimenti sugli animali descritti sono stati approvati dall'Institutional Animal Care and Use Committee del Boston Children's Hospital (protocollo ARCH numero 19-04-3913R).
1. Preparazione
<ol><li>Aggiungere 5 mL di penicillina/streptomicina (10000 U/mL) e 5 mL e 10 mL di integratori disponibili in commercio a 500 mL di mezzo classico completo con siero. Aliquota 50 mL del mezzo inizialmente. NOTA: Non restituire alcun mezzo al magazzino per evitare contaminazioni.</li>
	<li>Metti un'aliquota di mezzo classico completo sul ghiaccio.</li>
	<li>Utilizzare il 70% di etanolo per pulire il microscopio sezionato, le forcep e le forbici.</li>
	<li>Preparare due piatti di coltura cellulare (10 cm), uno al microscopio a dissezione, uno sul ghiaccio; mettere 10 mL di mezzo classico completo in ogni piatto.</li>
</ol>2. Fasi sperimentali(figura 1)
<ol><li>Sacrificare topi C57BL/6J intorno al postnatale (P) 20 usando 75-100 mg/kg di chetamina e 7,5-10 mg/kg di xiazina iniettata per via intraperitoneale. Tieni gli occhi in completo mezzo classico sul ghiaccio prima della dissezione.</li>
	<li>Rimuovere il tessuto connettivo (muscolo e tessuto adiposo) e il nervo ottico sull'occhio.</li>
	<li>Utilizzare una microforbice per tagliare circonferenzialmente 0,5 mm posteriore al limbo corneale. Rimuovere il complesso cornea/iris, vitreo e l'obiettivo.</li>
	<li>Rendere un'incisione di 1 mm perpendicolare al bordo tagliato verso il nervo ottico e tagliare una banda circonferenziale di 1 mm di larghezza. Separare le regioni centrali e periferiche del complesso. Utilizzare le forcep per staccare la retina dal complesso RPE/coroide/sclera.</li>
	<li>Mantenere la fascia coroide periferica in un mezzo classico completo sul ghiaccio; isolare l'altro occhio e ripetere il processo per tagliare una seconda fascia.</li>
	<li>Tagliare la banda circolare in 6 ~pezzi quadrati uguali (~1 mm x 1 mm). NOTA: non toccare mai alcun bordo.</li>
	<li>Scongelare l'estratto di membrana basale (BME) per istruzione della fabbricazione. Aggiungere 30 μL/pozzo di BME al centro di ogni pozzo di una piastra di coltura tissutale di 24 pozzo. Assicurarsi che la goccia di BME formi una cupola convessa nella parte inferiore della piastra senza toccare i bordi. NOTA: Scongelare il BME durante la notte in frigorifero. La BME deve essere sul ghiaccio in qualsiasi momento dopo lo scongelamento.</li>
	<li>Posizionare il tessuto al centro del BME. NOTA: Non appiattire l'espianto coroide; generalmente, lasciare che il tessuto si espanda all'interno del BME. L'orientamento del tessuto (lato sclerale verso l'alto o verso il basso) non ha un impatto sul risultato sperimentale.</li>
	<li>Incubare la piastra a 37 °C per 10 minuti per lasciare solidificare il gel.</li>
	<li>Aggiungere 500 μL del mezzo/pozzo classico completo.</li>
	<li>Cambia il mezzo classico a giorni diversi (500 μL). La germinazione coroide può essere osservata dopo 3 giorni con un microscopio. NOTA: Per il trattamento del fattore di crescita, morire di fame nel tessuto per 4 ore. Diluire un composto di prova nel mezzo ridotto dal fattore di crescita (aumento 1:200 invece di 1:50).</li>
</ol>3.SWIFT di quantificazione computerizzata a coroide17 (Figura 2)
NOTA: È stato utilizzato un metodo informatizzato per misurare la superficie coperta dai pescherecci in crescita. È necessario un plug-in macro per il software ImageJ prima della quantificazione (per ulteriori dettagli, vedere Informazioni supplementari).
<ol><li>Aprite l'immagine che germoglia coroide con ImageJ e controllate "Image | Tipo| 8 bit" con scala di grigi.</li>
	<li>Vai a "Image | Regolare | Luminosità/Contrasto (CTRL/MAIUSC/C)" e ottimizzare il contrasto.</li>
	<li>Utilizzare la funzione bacchetta magica per delineare e rimuovere dall'immagine il tessuto coroideo presente al centro dei germogli (usando il tasto di scelta rapida "F1") (Figura 2A,B). NOTA: Imposta il tasso di tolleranza della bacchetta magica sul 20-30%.</li>
	<li>Rimuovere lo sfondo dell'immagine con gli strumenti di selezione gratuiti (Figura 2C). Vai a "Image | Regolare | Soglia (CTRL/MAIUSC/T)". Utilizzare la funzione soglia per definire i germogli microvascolari sullo sfondo e sulla periferia (Figura 2D).</li>
	<li>Fare clic su "F2" e verrà visualizzato un riepilogo. Salvare un'immagine dell'area selezionata facendo clic su "Salva". Salvare nella stessa cartella dell'immagine originale per riferimento futuro.</li>
	<li>Dopo aver misurato un gruppo di campioni, copiare il registrato per l'analisi dei dati. NOTA: È anche possibile misurare l'area (μm²) da "Analizza | Set Scale" utilizzando immagini con barre di scala.</li>
</ol>

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F., Zorzi, P., Ligresti, G., Morishita, A., Aplin, 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=Paracrine+regulation+of+angiogenesis+by+different+cell+types+in+the+aorta+ring+model.">Paracrine regulation of angiogenesis by different cell types in the aorta ring model.</a> International Journal of Developmental Biology. 55 (4-5), 447-453 (2011).</li><li>Bellacen, K., Lewis, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+ring+assay.">Aortic ring assay.</a> Journal of Visulaized Experiments. (33), e1564 (2009).</li><li>Masson, V. 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H., 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=Endothelial+microRNA-150+is+an+intrinsic+suppressor+of+pathologic+ocular+neovascularization.">Endothelial microRNA-150 is an intrinsic suppressor of pathologic ocular neovascularization.</a> Proceedings of the National Academy of Science U. S. 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Gli autori non hanno informazioni finanziarie. Il metodo informatizzato è disponibile gratuitamente per gli istituti accademici attraverso gli autori.

Il saggio di germinazione coroidea aiuta la ricerca in AMD neovascolare9,10,18,19,20. Le espianto coroide possono essere isolate dai topi, dai ratti e dagli esseriumani 17,21. L'espianto coroide comprende EC, macrofagi e periciti17. In questo saggio l'interazione tra ec coroidia...

La disregolazione dell'angiogenesi coroiroidale è associata alla degenerazione maculare neovascolare legata all'età (AMD)1. La coroide è un letto microvascolare presente sotto l'epitelio pigmentato retinico (RPE). È stato dimostrato che il flusso sanguigno ridotto nella coroide è associato alla progressione dell'AMD2. L'intricata relazione tra endotelio vascolare, RPE, macrofagi, periciti e altre cellule è responsabile dell'omeostasi del tessuto3,4,5. Pertanto, un microambiente coroideo riproducibile che modella il test è fondamentale per lo studio dell'AMD neovascolare.
I test di angiogenesi ex vivo e le colture cellulari endoteliali in vitro possono integrare gli studi sul comportamento microvascolare in vivo, per testare nuovi farmaci e per studi di patogenesi. Cellule endoteliali come le cellule endoteliali microvascolari retiniche umane (HRMEC), le cellule endoteliali della vena ombelicale umana (HUVEC), il cervello animale primario isolato o gli EC retinali sono spesso utilizzati in studi in vitro per la ricerca sull'angiogenesi oculare6,7,8. Gli HRMEC in particolare sono stati ampiamente utilizzati come modello di neovascolarizzazione coroiroidale in vitro (CNV)9 valutando la proliferazione endoteliale, la migrazione, la formazione tubolare e le perdite vascolari pervalutare gli interventi 6,10. Tuttavia, gli EC in coltura sono limitati come modello di CNV a causa della mancanza di interazioni con altri tipi di cellule presenti nel coroide e perché la maggior parte della CE utilizzata in questi saggi non ha origine dalla coroide. Gli EC coroidiali del topo sono difficili da isolare e mantenere in coltura.
Il saggio dell'anello aortico è ampiamente usato come modello di proliferazione macrovascolare. I germogli vascolari provenienti da espianto aortico includono ESC, periciti e macrofagi11. Il saggio dell'anello aortico modella bene l'angiogenesi del vasodi grandi dimensioni 12,13,14. Tuttavia, ha dei limiti come modello di neovascolarizzazione coroidea in quanto gli anelli aortici sono un tessuto macrovascolare privo del caratteristico ambiente microvascolare coroiroidale, e i germogli di grandi vasi possono differire dai germogli dalle reti capillari coinvolte nella patologia microvascolare. Recentemente un gruppo ha pubblicato un saggio retinale ex vivo15,16. Sebbene sia adatto per la malattia neovascolare retinica, non è appropriato per la neovascolarizzazione coroiolare come si vede nell'AMD.
Il saggio di germinazione coroidea utilizzando RPE del topo, coroide e tessuto sclerale espiantato è stato sviluppato per modellare meglio la CNV. Il tessuto può essere facilmente isolato dagli occhi di topo (o altre specie)17. Questo saggio consente una valutazione riproducibile del potenziale pro e anti-angiogenico dei composti farmacologici e la valutazione del ruolo di percorsi specifici nella neovascolarizzazione coroiroidale utilizzando tessuti di topi geneticamente modificati econtrolli 18. Questo saggio di germinazione coroidea è stato citato in molte pubblicazioni successive9,10,18,19,20. Qui viene dimostrato il metodo coinvolto nell'uso di questo saggio.

<table>
	<tbody>
		<tr>
			<td>AnaSed (Xylazine)</td>
			<td>AKORN</td>
			<td>59339-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal membrane extract (BME) Matrigel</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td>10cm</td>
		</tr>
		<tr>
			<td>Complete classic medium with serum and CultureBoost</td>
			<td>Cell systems</td>
			<td>4Z0-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 Proof</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td>use for 70%</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEISS</td>
			<td>Axio Observer Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>GIBCO</td>
			<td>15140</td>
			<td>10000 U/mL</td>
		</tr>
		<tr>
			<td>Tissue culture plate (24-well)</td>
			<td>Olympus</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr>
			<td>VetaKet CIII (Ketamine)</td>
			<td>AKORN</td>
			<td>59399-114-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo choroid sprouting assay of ocular microvascular angiogenesis

L'angiogenesi coroiroidale patologica, una caratteristica saliente della degenerazione maculare legata all'età, porta a compromissione della vista e cecità. I test di proliferazione delle cellule endoteliali (CE) che utilizzano cellule endoteliali microvascolari retiniche umane (HRMEC) o EC retiniche primarie isolate sono ampiamente utilizzati modelli in vitro per studiare l'angiogenesi retinica. Tuttavia, isolare le cellule endoteliali retiniche murine pure è tecnicamente impegnativo e gli EC retinali possono avere risposte di proliferazione diverse rispetto alle cellule endoteliali coroidiali e alle diverse interazioni cellula/cellula. È stato sviluppato un saggio di germinazione coroidea ex vivo altamente riproducibile come modello di proliferazione microvascolare coroiroidale. Questo modello include l'interazione tra vascucolatura coroide (EC, macrofagi, periciti) e epitelio pigmentato retinico (RPE). Gli espianto di topo RPE/coroide/sclerale sono isolati e incubati nell'estratto di membrana basale (BME) ridotto dal fattore di crescita (giorno 0). Il mezzo viene cambiato a giorni diversi e la germinazione coroide viene quantificata al giorno 6. Le immagini dei singoli espianto coroide vengono scattate con un microscopio a fase invertita e l'area di germogliazione viene quantificata utilizzando un plug-in macro semi-automatizzato al software ImageJ sviluppato in questo laboratorio. Questo saggio di germinazione coroidea ex vivo riproducibile può essere utilizzato per valutare i composti per il potenziale trattamento e per la ricerca sulle malattie microvascolari per valutare le vie coinvolte nella proliferazione dei micro recipienti coroidiali utilizzando tipi selvatici e tessuti di topo geneticamente modificati.

Confronto della crescita della germogliazione coroide al giorno
Abbiamo sezionato la coroide con sclera, incorporata nel BME e coltivata per 6 giorni(Figura 1). La coroide che germoglia nei topi C57BL/6J dal giorno 3 al giorno 6 è stata esaminata con un microscopio e quantificata con SWIFT-Choroid un metodo di quantificazione semi-automatizzato in ImageJ. In un caso rappresentativo, la superficie di germogliazione coroioidea (i recipienti che si ...

Watch this Scientific Journal Video about An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis at JoVE.com

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Questo protocollo presenta il saggio di germinazione coroide, un modello ex vivo di proliferazione microvascolare. Questo saggio può essere utilizzato per valutare le vie coinvolte nei micro vasi cororoidali proliferano e valutare i trattamenti farmacologici utilizzando tipi selvatici e tessuti di topo geneticamente modificati.

Un test di germinazione coroide ex vivo di angiogenesi microvascolare oculare

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

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Research

JoVE Journal

Biology

5.5K Views.  Boston Children's Hospital, Harvard Medical School. Questo protocollo presenta il saggio di germinazione coroide, un modello ex vivo di proliferazione microvascolare. Questo saggio può essere utilizzato per valutare le vie coinvolte nei micro vasi cororoidali proliferano e valutare i trattamenti farmacologici utilizzando tipi selvatici e tessuti di topo geneticamente modificati.

Video: An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes: un modello in vitro per lo studio dell&#39;angiogenesi (Intervista)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

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Biologia

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Ottimizzato fibrina Gel Bead Assay per lo studio della angiogenesi

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Ex vivo Carico meccanico del tendine

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

Rat mesentere angiogenesi Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Endoteliali Tubo cellulare saggio Formazione per la<em&gt; In Vitro</em&gt; Studio di Angiogenesi

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

An Ex vivo Culture System to Study Thyroid Development

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 produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

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 ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

La proteina C-FOS immunoistologiche Detection: uno strumento utile come indicatore di percorsi centrali coinvolti nelle risposte fisiologiche specifiche<em&gt; In Vivo</em&gt; e<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

Un modello di cultura del tessuto Ex Vivo di Cartilage Rimodellamento in Esplants bovina del ginocchio

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Escrescenza glomerare come un'analisi di Ex Vivo per analizzare i percorsi coinvolti nell'attivazione delle cellule epiteliali Parietali

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Test di coltura di una singola miofibra per la valutazione della funzionalità delle cellule staminali muscolari adulte Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...