Questa ricerca è sostenuta dalla Fondazione Hartwell, l&#39;American Heart Association (Scientist Development Grant # 0830384N) e la Fondazione Leducq: 07CVD04 mitrale.

1. Preparazione <ol><li> Incubare uova di quaglia fertile o pollo allo stadio 14 - (circa 2 giorni) o 25 (circa 4,5 giorni) a 60% di umidità e 37 ° C. </li><li> Preparare la soluzione Balanced Salt Earl è sterile (EBSS) <ol><li> Aggiungere 100 mL EBSS 10X a 600 ml di acqua 18 MΩ </li><li> Aggiungere 2,2 g di sodio bicarbonato </li><li> Regolare il pH della soluzione a 7,2 </li><li> Portare la soluzione fino a 1000 ml </li><li> Sterilizzare la soluzione passa attraverso un filtro 0,2 micron </li></ol></li><li> Preparare sterile M199 terreno di coltura. <ol><li> Aggiungere 1 confezione di M199 in polvere a 700 ml di acqua 18 MΩ. </li><li> Aggiungere 2,2 g di sodio bicarbonato </li><li> Regolare il pH della soluzione a 7,2. </li><li> Aggiungere 10 ml (1%) Penicillina-Streptomicina </li><li> Portare la soluzione fino a 1000 ml </li><li> Sterilizzare la soluzione passa attraverso un filtro 0,2 micron </li><li> Aggiungere 1 ml sterile 100X Insulin-transferrina-selenio-G supplemento </li><li> Aggiungere 10 ml (1%) siero di pollo sterile </li></ol></li><li> Preparare tre gel tridimensionale di collagene ad una concentrazione di collagene di 1,5 mg / ml. Lavoro precedente nel nostro laboratorio ha dimostrato che una concentrazione di collagene di più di 1,5 mg / ml fornisce una matrice che è troppo rigida biomeccanico per le cellule per spostarsi. Concentrazioni di gel di collagene inferiore a 1,5 mg / ml sono fibre di collagene così pochi che il ciuffo cellula può distruggere le fibre a livello locale, producendo un &#39;isola di cellule. <ol><li> Aggiungere la seguente ghiacciata reagenti ad una provetta sterile 15 ml in ordine: <ul><li> 3X M199: Volume = volume totale necessario / 3 </li><li> 18 MΩ acqua sterile: Volume = volume totale necessario (M199 volume + volume di pollo siero + 0,1 M di NaOH il volume + volume collagene) </li><li> Siero di pollame sterile: Volume = volume totale necessario * 0.01 </li><li> Rat collagene coda I: Volume = (gel di concentrazione di collagene * volume totale necessario) / collagene concentrazione magazzino </li><li> Sterile 0,1 M NaOH: Volume = volume collagene * 0,2 Nota: La quantità di acqua, 0,1 M di NaOH, e collagene aggiunto varierà a seconda della concentrazione della soluzione di riserva collagene. </li></ul></li><li> Miscelare la soluzione collagene bene con una pipetta sterile. </li><li> Trasferire immediatamente il 0,3 mL della soluzione di collagene in 1,9 cm 2 pozzi di crescita dell&#39;area. Questa è la soluzione di collagene sufficiente a coprire completamente il bene e per fornire un gel che è di circa 3 mm di spessore. Un gel spessore di almeno 250 micron è necessario per gli studi di invasione delle cellule. </li><li> Lasciare che la soluzione di collagene gel per almeno 1 ora a 37 ° C, 5% CO 2, e 100% di umidità. </li></ol></li></ol> 2. Pre-EMT endocardica isolamento delle cellule e Cultura <ol><li> Embrione isolamento <ol><li> Posizionare un pezzo pollice di lunghezza del nastro di laboratorio oltre l&#39;estremità più grande di un uovo. Questo è dove la cellula d&#39;aria si trova. Il nastro stabilizza il fragile guscio d&#39;uovo di quaglia. </li><li> Delicatamente forare la superficie registrata dell&#39;uovo nella cella d&#39;aria con la punta delle forbici curve sterilizzati. </li><li> Praticare un foro nella parte registrata dell&#39;uovo che è grande abbastanza per permettere il tuorlo di passare attraverso. Avviare il foro alla puntura e tagliare verso l&#39;esterno a forma di spirale. Assicurati di tagliare la membrana esterna, ma evitare di forare il tuorlo. </li><li> Quando il buco nella shell è completo, visualizzare l&#39;embrione di quaglia. L&#39;embrione è normalmente situato sul lato superiore del tuorlo. </li><li> Cominciate a versare il tuorlo attraverso il taglio foro nell&#39;uovo con una mano. Tenere un # 55 pinzetta nell&#39;altra mano. Come l&#39;embrione esce l&#39;uovo sul lato superiore del tuorlo, scorrere delicatamente il # 55 pinzette sotto l&#39;embrione e rimuoverlo dal tuorlo. Nota: per uova di gallina, l&#39;isolamento embrione comporta rottura le uova di pollo su un forte, superficie pulita e rompere l&#39;uovo in un piatto da 100 mm di Petri. L&#39;embrione deve essere posizionato sul lato superiore del tuorlo. Far scorrere delicatamente # 55 pinzette sotto l&#39;embrione e rimuoverlo dal tuorlo. </li><li> Mettere l&#39;embrione in un piatto 100 mm Petri contenenti ghiacciata, EBSS sterile. </li></ol></li><li> Fase gli embrioni secondo i criteri di Hamburger e Hamilton 1. Embrioni allo stadio 14 - sono stadio ultimi 13, ma non ancora in fase di 15, con 20-21 somiti ed una gemma coda. Una caratteristica chiave della tappa 14 - embrioni è l&#39;angolo di testa, che è leggermente superiore a 90 ° rispetto al corpo. </li><li> Rimuovere i cuori da embrioni di quaglia allo stadio 14 - con N ° 55 pinzette, tagliando trasversalmente in cui il canale AV e OFT entrare in contatto con la parete toracica. Mettere i cuori insieme su un lato della piastra di Petri. </li><li> Raccogliere i cuori isolati con una pipetta sterile per il trasferimento e metterli in un piatto nuovo 100 millimetri Petri riempite con una soluzione sterile, ghiacciata EBSS. </li><html><li> Tagliare il OFTs e canali AV trasversalmente dai ventricoli con # 55 pinzette. </li><li> Sezione i canali restanti AV e / o vie di deflusso (OFTs) longitudinalmente. </li><li> Impostare una pipetta ad un volume di 20 uL. Utilizzare questa pipetta per aspirare sei tagliato longitudinalmente canali AV e OFTs. </li><li> Espellere i canali AV e OFTs sul gel di collagene. Aspirare qualsiasi EBSS eccesso dalla superficie del gel. </li><li> Utilizzare # 55 pinzette per orientare la espianti in modo che siano piatte e quindi il lume lato si affaccia sul gel di collagene. Cercate di non forare il gel di collagene con le pinzette. </li><li> Dopo 2 ore a 37 ° C e 5% di CO 2, rimuovere il miocardio dal espianti con # 55 pinzette. Le cellule endocardico dovrebbe essere lasciato sulla superficie del gel di collagene. In alternativa, il miocardio può essere lasciato sul espianto. Con la rimozione miocardio, le cellule endocardici con non sottoposti EMT senza alcun intervento. Con il miocardio presente un sottogruppo di cellule endocardico si trasformerà in mesenchima, ma il grado di trasformazione può essere modulata da fattori esterni. Nota: Anche se il miocardio sarà lasciato, l&#39;espianto deve essere consentito almeno 2 ore da allegare al gel prima media viene aggiunto. </li><li> Aggiungere 0,4 ml M199 medio pozzetti contenenti ciascuno espianti. </li><li> Coltura le cellule a 37 ° C, 5% CO 2, e 100% di umidità. </li></ol> 3. Post-EMT AV Cuscino isolamento delle cellule mesenchimali e Cultura <ol><li> Rompere le uova di pollo su un forte, superficie pulita e rompere l&#39;uovo in un piatto da 100 mm di Petri. L&#39;embrione deve essere posizionato sul lato superiore del tuorlo. Raccogliete l&#39;embrione con # 5 pinzette afferrando membrane che circondano l&#39;embrione e poi posto l&#39;embrione in un piatto 100 millimetri Petri riempite con ghiaccio freddo, EBSS sterile. </li><li> Fase gli embrioni secondo i criteri di Hamburger e Hamilton 1. Caratteristiche di una fase embrionale 25 sono distinte gomito e ginocchio e la pigmentazione degli occhi leggermente. </li><li> Isolare i cuori di tutti HH25 embrioni e il luogo del cuore insieme su un lato della piastra di Petri. </li><li> Raccogliere i cuori isolati con una pipetta sterile per il trasferimento e metterli in un piatto nuovo 100 millimetri Petri riempite con una soluzione sterile, ghiacciata EBSS. </li><li> Isolare la regione AV da tutti i cuori in una volta. L&#39;AVS posto insieme su un lato della piastra di Petri. Agitare delicatamente la regione AV per rimuovere tutto il sangue, che può influenzare la vitalità cellulare. </li><li> Raccogliere il isolato AVS con una pipetta sterile per il trasferimento e metterli in un piatto nuovo 100 millimetri Petri riempite con una soluzione sterile, ghiacciata EBSS. </li><li> Sequenziale isolare i cuscini dal AV. Assicurarsi che non sia presente miocardio sui cuscini AV. </li><li> Raccogliere i cuscini isolati con una pipetta sterile per il trasferimento e metterli in una provetta sterile 15 ml per centrifuga. </li><li> Spin i cuscini sul fondo della provetta da centrifuga a 15 xg per 3 minuti. </li><li> Utilizzare una punta sterile 1000 ml per rimuovere la maggior quantità di surnatante EBSS possibile senza disturbare i cuscini. </li><li> Aggiungere 1 ml di 0,25% tripsina-EDTA che è stato pre-riscaldato a 37 ° C. Lasciare i cuscini ad incubare in tripsina fino a quando sono completamente sciolti, che normalmente è di circa 3-5 minuti. </li><li> Aggiungere 100 ml di siero di pollo al tubo di centrifuga per spegnere la tripsina. </li><li> Agglomerare le cellule a 160 xg per 5 minuti. Eliminare il surnatante con una sterile, punta di 1000 ml. </li><li> Risospendere le cellule in 2 ml di medium M199 e mescolare pipettando. Prendere 10 microlitri della sospensione cellulare contare su emocitometro e determinare il numero totale di cellule che sono state isolate </li><li> Determinare il numero di gel di collagene verrà effettuato in base al numero di cellule isolate. Celle devono essere placcati con una densità di 2 x 10 5 cellule / ml con un volume di 250 microlitri di gel. </li><li> Agglomerare le cellule a 160 xg per 5 minuti. Eliminare il surnatante con una sterile, punta di 1000 ml. </li><li> Utilizzando il protocollo di cui sopra, aggiungere 3X M199, acqua, pollo siero, collagene, e 0,1 M di NaOH, in questo ordine, al pellet. Mescolare pipettando gentile. </li><li> Aggiungere 250 microlitri per ogni cm 1,9 2 area di crescita bene. </li><li> Lasciare che la soluzione di collagene gel per almeno 1 ora a 37 ° C, 5% CO 2, e 100% di umidità. </li><li> Aggiungere 0,4 ml di terreni di coltura e incubare una notte. </li><li> Per continuare a coltivare il gel in condizioni di stress, liberare il gel dai lati della cultura e con una sterile 200 microlitri punta della pipetta. Culture meccanicamente vincolato sono gel che rimangono attaccati ai lati della cultura del bene. </li></ol> 4. Rappresentante Risultati <img src="/files/ftp_upload/2159/2159fig1.jpg" alt="Figura 1" /> Figura 1:. Esempi di embrioni (A) HH14-quaglia embrione, e (B) HH25 embrione di pollo sono mostrati qui. fig2.jpg &quot;alt =&quot; Figura 2 &quot;/&gt; Figura 2: HH14 valvole espianti endocardico dopo 2 ore di cultura (A) con il presente miocardio, o (B) con il miocardio rimosso.. <img src="/files/ftp_upload/2159/2159fig3.jpg" alt="Figura 3" /> Figura 3:. HH14 valvole espianti endocardico (A) Con la presente miocardio, molte delle cellule subiscono endocardica alla trasformazione mesenchimali ed avere un mandrino a forma di fenotipo mesenchimale dopo 48 ore di cultura. (B) Quando il miocardio è stato rimosso tutte le cellule mantenere una forma di ciottoli, fenotipo endocardico dopo 48 ore di cultura. <img src="/files/ftp_upload/2159/2159fig4.jpg" alt="Figura 4"/> Figura 4:. HH25 valvola cellule mesenchimali in un gel di collagene Le cellule sono arrotondati subito dopo la semina. <img src="/files/ftp_upload/2159/2159fig5.jpg" alt="Figura 5"/> Figura 5:. HH25 cellule mesenchimali in coltura (A) Dopo 48 ore di cultura in un gel di collagene limitato la HH25 cellule mesenchimali stanno cominciando a diffondersi. (B) HH25 cellule in uno stress senza gel di collagene 7 giorni dopo la semina e 6 giorni dopo la liberazione hanno compattato il gel a circa il 50% della superficie originale.

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88, 49-92 (1951).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Biology. 95, 108-108 (1983).</li><li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> Developmental Biology. 302, 256-256 (2007).</li></ol>

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Il metodo per isolare le cellule endocardico da stadio HH14 - Cuori originariamente sviluppato da Runyan e Markwald fornisce una controllata, in ambiente vitro per studiare i fattori che innescano e modulano embrionale EMT 2. HH14 - espianti endocardico colta senza miocardio non subirà EMT senza l&#39;intervento biomeccanici o biochimici. Se il miocardio è lasciato sul espianto sarà il segnale di un sottogruppo di cellule endocardico a subire la trasformazione mesenc...

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<td>Extra fine Bonn scissors, curved</td>
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<td>Fine Science Tools</td>
<td>14085-08</td>
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<td>Dumont tweezers #55</td>
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<td>Sterile transfer pipettes</td>
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<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
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<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
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<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
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<td>Nalgene</td>
<td>566-0020</td>
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<td>Sterile 15 mL centrifuge tubes</td>
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<td>Sterile petri dishes, 100 mm x 15 mm</td>
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<td>Rat-tail collagen I</td>
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<td>10X Earl's Balanced Salt Solution</td>
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<td>Sigma-Aldrich</td>
<td>S2770</td>
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<td>Fertile quail eggs (Coturnix coturnix)</td>
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<td>Lake Cumberland Game Bird Farm</td>
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<td>Fertile chicken eggs (Gallus gallus)</td>
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<td>Cornell University Poultry Farm</td>
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isolation and culture of avian embryonic valvular progenitor cells

Un&#39;adeguata formazione e la funzione delle valvole cardiache embrionali è fondamentale per la progressione di sviluppo. Il cuore embrionale precoce è un tubo a U di endocardio circondato da miocardio. Miocardio secerne pappa cardiaci, un acido ialuronico ricco di matrice gelatinosa, nel nodo atrioventricolare (AV) e del tratto di efflusso (OFT) lumen. Nella fase HH14 valvulogenesis inizia quando un sottogruppo di cellule endocardico ricevere segnali dai miocardio, sottoposti endocardico alla trasformazione mesenchimale (EMT), e invadono la pappa cardiaci. Nella fase HH25 i cuscini sono completamente valvolare mesenchymalized, ed è questo mesenchima che forma poi l&#39;apparato valvolare e del setto del cuore. La comprensione dei meccanismi che innescano e modulano il processo di differenziazione delle cellule EMT e sono importanti per la loro connessione con gravi difetti cardiaci congeniti. In questo studio presenteremo i metodi per isolare pre-EMT endocardico e post-EMT cellule mesenchimali, che sono le due fenotipi cellulari diversi del cuscino prevalvular. Pre-EMT cellule endocardico possono essere coltivate con o senza il miocardio. Post-EMT cuscino AV cellule mesenchimali possono essere coltivate gel di collagene all&#39;interno meccanicamente vincolato o senza stress. Queste 3D in modelli in vitro imitare principali eventi morfogenici valvolare e sono utili per decostruire i meccanismi di valvulogenesis fase precoce e tardiva.

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Questo articolo fornisce un metodo per isolare e quaglia coltura o pollo HH14<sup&gt; -</sup&gt; Valvola di cellule endocardico e HH25 cellule mesenchimali cuscino valvola.

Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

isolation-and-culture-of-avian-embryonic-valvular-progenitor-cells

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Biology

10.1K Views.  Cornell University. Questo articolo fornisce un metodo per isolare e quaglia coltura o pollo HH14 - Valvola di cellule endocardico e HH25 cellule mesenchimali cuscino valvola.

Video: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

Isolamento e la coltura di cellule post-Natal Neuron cerebellare granuli mouse progenitore e Neuroni

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Ricerca

Biologia

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Cultura e manutenzione di cellule staminali embrionali umane

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolamento e di espansione su vasta scala di umano adulto cellule progenitrici endoteliali che formano colonie

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Isolamento di cellule endoteliali valvolare

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e coltura di cellule dalla Zona nefrogenica del Rene embrionali di topo

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolamento e la coltura primaria di cellule epatiche di ratto

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolamento della embrionali ventricolari cellule endoteliali

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...