Il finanziamento per lo sviluppo del protocollo attuale è sostenuta in particolare da &quot;Obiettivo per il Piano Top Università&quot; della National Taiwan University, Taiwan (grant ID-104R350144), così come il Ministero della Scienza e della Tecnologia di Taiwan (grant ID: PIU &#39;104 -2313-B-002-039-MY3). Ringraziamo in modo particolare il Centro di Biotecnologie della National Taiwan University per fornire un ambiente animale e spazio laboratorio per lo studio corrente.

NOTA: Questa procedura è una modifica di un modello di pollo protocollo di cultura sviluppato da Bauer et al 2013 e Nakazawa et al, 2011. 2,9.. 1. Preparare sano embrionale Giorno 5 embrioni da Quail giapponese <ol><li> Mettere 1 maschio e 3 femmine sessualmente maturi quaglia giapponese insieme nella stessa gabbia. Portare l&#39;alimentazione e acqua ad libitum. Regolare la luce nella stanza animale 14 ore di luce e 10 ore di buio. </li><li> Raccogliere le uova fecondate in sacchetti di plastica ogni giorno nel pomeriggio e tenerli in un frigorifero C 16 ° per ritardare il tasso di crescita degli embrioni. </li><li> Dopo aver raccolto un numero sufficiente di uova in un periodo di due settimane, incubare le uova fecondate in un incubatore a 37 ° con una buona ventilazione per cinque giorni. </li></ol> 2. Preparare basale medio e Wash Solutions <ol><li> Utilizzare DMEM / F-12 di media (regolata a pH 7.2) come mezzo di crescita, e integrarlacon il 10% nuovo siero vitello nato (siero bovino neonatale) e l&#39;1% Pen-Strep Ampho. Soluzione (PSA, penicillina, 10 5 unità / l; streptomicina, 100 mg / l; amfotericina B, 0,25 mg / L). </li><li> Preparare la soluzione tampone fosfato (PBS) ad una concentrazione 10 volte per lo stoccaggio; usare una soluzione di PBS 1x (contenente NaCl, 137.93 mm, KCl, 2.667 mm, KH 2 PO 4, 1.471 mm, Na 2 HPO 4 -7h 2 O, 8,06 mm, regolare a pH 7,2) per lavare e inumidire il sacco vitellino membrana mentre che separa il endoderma dal tuorlo d&#39;uovo e l&#39;albumina. </li><li> Utilizzare terreno DMEM (regolata a pH 7,2) per risospendere pellet endoderma. </li></ol> 3. Raccolta di primaria TEE da Quail YSMs giapponesi <ol><li> Incubare le uova raccolte per 5 giorni (passo 1,3). Esaminare le uova da parte di un Candler uovo per confermare il normale sviluppo embrionale. NOTA: Il embrionali giorno 5, il confine del YSM mesoderma è molto chiara e le 3 strati embrionali sono meno strettamente collegati tra loro, per cui è facile da obTain endoderma. Utilizzare solo le uova che mostrano lo sviluppo normale circolazione capillare per le collezioni YSM. </li><li> Pulire il guscio d&#39;uovo accuratamente con acqua fresca e il 75% di etanolo. Aprire il guscio d&#39;uovo dalla posizione di cella d&#39;aria per un paio di forbici, delicatamente sbucciare e raccogliere tutta la YSM da pinze e posto in un piatto della cultura 10 centimetri pieno di 37 ° C 1x PBS. </li><li> Utilizzando un paio di forbici rimuovere l&#39;embrione, tuorlo e albume d&#39;uovo, e delicatamente lavare la YSM con 1x PBS tre volte e posizionare YSM in soluzione PBS in un piatto della cultura 10 cm. </li><li> Utilizzare pinze e un paio di forbici per rimuovere il ectoderma da YSM. Utilizzare 2 pinze, una per tenere saldamente lo strato di cellule endodermico, e l&#39;altro per tenere il mesoderma capillare. </li><li> Sotto un microscopio da dissezione, separare e separare l&#39;endoderma dal bordo del mesoderma nella direzione verso l&#39;embrione. Raccogliere il endoderma giallo chiaro in 50 ml provette da centrifuga con un contagocce e posto in 15 ml di mezzo di coltura in un C wat 37 °er bagno fino a tutte le collezioni di tessuti sono stati completati. </li></ol> 4. La digestione del Endoderma fette da digestione collagenasi <ol><li> Appena sciogliere 6,5 unità di tipo IV collagenasi in 10 ml di DMEM. </li><li> Centrifugare le provette da 50 ml (3.4) a 130 xg per 3 min a RT. Aspirare il terreno e mettere tutto il endoderma in un piatto di cultura 6 centimetri con una pipetta. Aggiungere la soluzione collagenasi 1 ml di questo piatto della cultura. </li><li> Tagliare il endoderma raccolto da 2 embrioni quaglia con un paio di forbici curve finché le dimensioni di ogni fetta è di circa 2 a 3 mm. Utilizzare un contagocce, raccogliere tutte le fette in un tubo da 50 ml centrifuga insieme a 9 ml di soluzione di collagenasi fresca. </li><li> Digerire il tessuto con collagenasi per 30 min a 37 ° C agitando bagnomaria a 175 rpm. Questo parziale digestione con collagenasi viene utilizzato per migliorare la proliferazione cellulare espianto. </li><li> Centrifugare a 130 xg per 3 minuti a temperatura ambiente e rimuovere delicatamente la frazione surnatante con una pipetta. Lavare il pellet mediante sospensione in 20 ml di DMEM e rimuovere il surnatante dopo 130 xg centrifugazione per 3 min. </li><li> Risospendere il pellet con 12 ml di terreno di coltura (DMEM / F12 con il 10% e l&#39;1% NBCS PSA) e seminare con delicatezza e altrettanto in un 24-pozzetti (ogni soluzione cella bene con 500 microlitri). </li><li> Incubare espianti di tessuto a 37 ° C in 5% CO 2 in aria per 2 giorni per consentire cellule a proliferare dai tessuti. </li><li> Incubare le cellule per altri 2 giorni e si caratterizza per la vitalità cellulare da un protocollo di test di vitalità cellulare. 15 NOTA: Living cellule mantengono un ambiente riducente all&#39;interno del citoplasma. Resazurina, l&#39;ingrediente attivo, è non tossico, cellule composto permeabile che è di colore blu e praticamente non fluorescente. Una volta aggiunto al colture cellulari, resazurina si riduce a resorufina nel citosol dagli enzimi mitocondriali. Resorufina è di colore rosso e altamente fluorescente. cellule vitali continuamente convertire resazurina a resorufina, increAsing la fluorescenza complessiva e il colore del mezzo di coltura delle cellule circostanti. </li><li> Eseguire endoderma cellule epiteliali isolamento RNA totale, trascrizione e quantitativa real-time PCR dell&#39;espressione dell&#39;mRNA gene marcatore invertire come descritto. 16,17 <ol><li> Utilizzare PCR trascrittasi inversa per rilevare SOAT1 mRNA (senso: 5&#39;-GAAGGGGCCTATCTGGAACG-3 &#39;; antisenso: 5&#39;-ATCTGCACGTGACATGACCA-3&#39;; PCR prodotto = 168 bp) come marcatore specifico per le cellule CEE. Utilizzare β-actina mRNA (senso: 5&#39;- TGGTGAAGCTGTAGCCTCTC-3 &#39;; antisenso: 5&#39;-GTGATGGACTCTGGTGATGG-3&#39;; prodotto di PCR = 151 bp) come gene housekeeping e di controllo interno. </li><li> Utilizzare elettroforesi su gel per rilevare la dimensione del prodotto di RT-qPCR. Preparare 1% gel, e mescolare 10 ml di prodotto qPCR con la tintura di carico 2 ml. Carico 10 microlitri miscela in ciascun pozzetto nel gel di agarosio 1%, ed eseguire il gel mediante 100V per 30 min. </li></ol></li></ol>

<ol>
	<li>Abeyrathne, E. D., Lee, H. Y., Ahn, D. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg+white+proteins+and+their+potential+use+in+food+processing+or+as+nutraceutical+and+pharmaceutical+agents--a+review.">Egg white proteins and their potential use in food processing or as nutraceutical and pharmaceutical agents--a review.</a> Poult Sci. 92 (12), 3292-3299 (2013).</li><li>Bauer, R., Plieschnig, J. A., Finkes, T., Riegler, B., Hermann, M., Schneider, W. J. <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+chicken+yolk+sac+acquires+nutrient+transport+competence+by+an+orchestrated+differentiation+process+of+its+endodermal+epithelial+cells.">The developing chicken yolk sac acquires nutrient transport competence by an orchestrated differentiation process of its endodermal epithelial cells.</a> J Biol Chem. 288 (2), 1088-1098 (2013).</li><li>Ding, S. T., Nestor, K. E., Lilburn, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concentration+of+different+lipid+classes+during+late+embryonic+development+in+a+randombred+turkey+population+and+a+subline+selected+for+increased+body+weight+at+sixteen+weeks+of+age.">The concentration of different lipid classes during late embryonic development in a randombred turkey population and a subline selected for increased body weight at sixteen weeks of age.</a> Poult Sci. 74 (2), 374-382 (1995).</li><li>Ding, S. T., Lilburn, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+expression+of+acyl-coenzyme+A:+cholesterol+acyltransferase+in+the+yolk+sac+membrane,+liver,+and+intestine+of+developing+embryos+and+posthatch+turkeys.">The developmental expression of acyl-coenzyme A: cholesterol acyltransferase in the yolk sac membrane, liver, and intestine of developing embryos and posthatch turkeys.</a> Poult Sci. 79 (10), 1460-1464 (2000).</li><li>Ding, S. T., Lilburn, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ontogeny+of+fatty+acid-binding+protein+in+turkey+(Meleagridis+gallopavo)+intestine+and+yolk+sac+membrane+during+embryonic+and+early+posthatch+development.">The ontogeny of fatty acid-binding protein in turkey (Meleagridis gallopavo) intestine and yolk sac membrane during embryonic and early posthatch development.</a> Poult Sci. 81 (7), 1065-1070 (2002).</li><li>Kanai, M., Soji, T., Sugawara, E., Watari, N., Oguchi, H., Matsubara, M., Herbert, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+endodermal+epithelial+cells+on+the+synthesis+of+plasma+LDL+and+HDL+in+the+chick+yolk+sac.">Participation of endodermal epithelial cells on the synthesis of plasma LDL and HDL in the chick yolk sac.</a> Microsc Res Tech. 35 (4), 340-348 (1996).</li><li>Mobbs, I. G., McMillan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+endodermal+epithelium+of+the+chick+yolk+sac+during+early+stages+of+development.">Structure of the endodermal epithelium of the chick yolk sac during early stages of development.</a> Am J Anat. 155 (3), 287-309 (1979).</li><li>Mobbs, I. G., McMillan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+across+endodermal+cells+of+the+chick+yolk+sac+during+early+stages+of+development.">Transport across endodermal cells of the chick yolk sac during early stages of development.</a> Am J Anat. 160 (3), 285-308 (1981).</li><li>Nakazawa, F., Alev, C., Jakt, L. M., Sheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yolk+sac+endoderm+is+the+major+source+of+serum+proteins+and+lipids+and+is+involved+in+the+regulation+of+vascular+integrity+in+early+chick+development.">Yolk sac endoderm is the major source of serum proteins and lipids and is involved in the regulation of vascular integrity in early chick development.</a> Dev Dyn. 240 (8), 2002-2010 (2011).</li><li>Noble, R. C., Cocchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+and+the+neonatal+chicken.">Lipid metabolism and the neonatal chicken.</a> Prog Lipid Res. 29 (2), 107-140 (1990).</li><li>Shand, J. H., West, D. W., McCartney, R. J., Noble, R. C., Speake, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+esterification+of+cholesterol+in+the+yolk+sac+membrane+of+the+chick+embryo.">The esterification of cholesterol in the yolk sac membrane of the chick embryo.</a> Lipids. 28 (7), 621-625 (1993).</li><li>Speier, J. S., Yadgary, L., Uni, Z., Wong, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+of+nutrient+transporters+and+digestive+enzymes+in+the+yolk+sac+membrane+and+small+intestine+of+the+developing+embryonic+chick.">Gene expression of nutrient transporters and digestive enzymes in the yolk sac membrane and small intestine of the developing embryonic chick.</a> Poult Sci. 91 (8), 1941-1949 (2012).</li><li>Walzem, R. L., Hansen, R. J., Williams, D. L., Hamilton, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+Induction+of+VLDLy+Assembly+in+Egg-Laying+Hens.">Estrogen Induction of VLDLy Assembly in Egg-Laying Hens.</a> J Nutr. 129 (2), 467S-472S (1999).</li><li>Yoshizaki, N., Soga, M., Ito, Y., Mao, K. M., Sultana, F., Yonezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+consumption+of+yolk+granules+during+the+development+of+quail+embryos.">Two-step consumption of yolk granules during the development of quail embryos.</a> Dev Growth Differ. 46 (3), 229-238 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> alamarBlue Cell Viability Assay Protocol. , (2008).</li><li>Lin, H. Y., Chen, C. C., Chen, Y. J., Lin, Y. Y., Mersmann, H. J., Ding, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Amelioration+of+High-Fat+Diet-Induced+Fatty+Liver+by+Docosahexaenoic+Acid+and+Lysine+Supplementations.">Enhanced Amelioration of High-Fat Diet-Induced Fatty Liver by Docosahexaenoic Acid and Lysine Supplementations.</a> Biomed Res Int. 2014 (1), 310981 (2014).</li><li>Lin, Y. Y., 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=Adiponectin+receptor+1+regulates+bone+formation+and+osteoblast+differentiation+by+GSK-3&#946;/&#946;-Catenin+signaling+in+mice.">Adiponectin receptor 1 regulates bone formation and osteoblast differentiation by GSK-3&#946;/&#946;-Catenin signaling in mice.</a> Bone. 64, 147-154 (2014).</li></ol>

There is no competing financial interests or other conflicts of interest. We did not get any funding from any private company.

Poiché il sistema di coltura precedente ha solo un successo limitato, è necessario un sistema di coltura migliore. L&#39;endoderma quaglie YSM giapponese richiede un trattamento con enzimi proteolitici, come collagenasi per allentare le giunzioni cellula-cellula per ottenere una migliore performance di crescita negli espianti ex-vivo. I nostri dati mostrano che il numero di cellule derivanti dal trattamento di digestione parziale sono stati superiori dalla coltura tissutale non digerito dopo la semina per 2 giorni <st...

La principale risorsa alimentare dell&#39;embrione aviaria è tuorlo, composto da 33% di lipidi, proteine ​​17% e 1% di ceneri. 1 Durante lo sviluppo embrionale, la membrana sacco vitellino (YSM) cresce all&#39;interno della cavità addominale embrionale e copre gradualmente il tuorlo superficie. Con inizio alle embrionali giorno 2, l&#39;espressione del gene associato con il metabolismo dei lipidi e l&#39;angiogenesi è gradualmente aumentata in YSM, e il YSM sviluppa lentamente proiezioni villi-like. 8,9 Queste proiezioni aumentano l&#39;assorbimento delle sostanze nutritive tuorlo per sostenere lo sviluppo embrionale. Il YSM è un tessuto extraembryonic che contiene tre strati germinali, endoderma, mesoderma ed ectoderma. 14 Il ectoderma sacco vitellino si trova di fronte l&#39;albume e collegamenti con la membrana vitellina per coprire delicatamente il sacco vitellino. Le cellule epiteliali endodermico si trovano ad affrontare direttamente verso il tuorlo d&#39;uovo e servono come portali di utilizzo di nutrienti. 6 Come si espande il YSM, endodermico cellule epiteliali (TEE) può essere diviso per ShapE e funzionalità in due gruppi, zona vitellina e zona vasculosa. 7 Area vitellina è composto da cellule endodermico ed è distante dalla dell&#39;embrione; zona vasculosa è composto da cellule mesodermiche e copre TEE differenziati con i vasi sanguigni e dei tessuti connettivi. Di embrionali giorno 5, il tuorlo è totalmente coperto da ectoderma e endoderma di YSM e l&#39;area vascolare è cresciuto rapidamente. Il YSM assorbe, ricompone e rilascia i lipidi (come il tuorlo di derivazione molto lipoproteine ​​a bassa densità) e le proteine ​​nel sistema circolatorio embrionale. 9, 2 Pertanto, abbiamo stabilito un embrionale sistema di coltura cellulare primaria giapponese quaglie endodermico epiteliale, per studiare i meccanismi di lipidi utilizzo in YSM durante lo sviluppo embrionale aviaria. I lipidi quali trigliceridi, lecitina, fosfolipidi e colesterolo estere (CE) sono le fonti di energia primaria per gli embrioni aviaria. Nelle prime fasi di sviluppo, lipidi tuorlo sono composed di solo l&#39;1,3% del CE e sale al 10-15% a medio termine della aviaria sviluppo embrionale 3, 11. estere Il colesterolo è sintetizzato dal colesterolo da steroli O-aciltransferasi 1 (SOAT1) in aviario YSM embrionale. 4 La forma di colesterolo stoccaggio è CE, CE viene effettuata in lipoproteine, e lipoproteine ​​vengono trasportati dalla circolazione ai tessuti. 13 Una settimana prima portello c&#39;è una rapida crescita degli embrioni aviaria. Circa il 68% delle restanti contenuti di lipidi nel tuorlo vengono assorbiti durante questa fase. 10 Il meccanismo con cui tuorlo lipidi vengono utilizzati possono essere chiarita da un modello di ricerca CEE. Un protocollo cultura pollo CEE è stato creato per raggiungere questo obiettivo di ricerca. 2, 9 Tuttavia, a causa del basso tasso di successo di espianti di tessuto, è necessario un procedimento di coltura cellulare CEE per studiare la funzione di alcuni enzimi e proteine ​​nel mediare utilizzazione nutriente embrioni aviaria durante lo sviluppo.

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12800-017 10 X 1 L</td>
			<td style="width:167px;">For wash the EECs pellets</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">D-MEM/F-12</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12400-024 10 X 1 L</td>
			<td>As the basal medium in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">NBCS</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">16010-159</td>
			<td>As the supplyment serum in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Pen-Strep Ampho. Solution</td>
			<td style="width:199px;">BI (Biological Industries)</td>
			<td style="width:148px;">03-033-1B 100ml</td>
			<td>For attenuating the possible infection&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Collagenase Type IV</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">17104-019 1g</td>
			<td>&#160;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">24 well plate</td>
			<td style="width:199px;">FALCON&#174;</td>
			<td style="width:148px;">REF-353047</td>
			<td>For EECs to attach and extension</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">50 ML PP centrifuge tubes</td>
			<td style="width:199px;">Corning&#174; CentriStarTM</td>
			<td style="width:148px;">430829</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">50ML Conical bottomed Tube with Cap</td>
			<td style="width:199px;">PRO TECH</td>
			<td>CT-50-PL-TW</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Reciprocal shaking bath</td>
			<td>DEAGLE</td>
			<td style="width:148px;">SB302</td>
			<td>For better enzymatic digestion on membranes</td>
		</tr>
	</tbody>
</table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

Abbiamo stabilito un endodermico epiteliale modello di coltura cellulare (CEE) per studiare la funzione di alcuni enzimi e proteine ​​nel mediare l&#39;utilizzo dei nutrienti da embrioni aviaria durante lo sviluppo. Fecondate uova di quaglia giapponesi sono state incubate a 37 ° C per 5 giorni e poi tuorlo membrane SAC (YSM) sono stati raccolti per stabilire il sistema di coltura CEE. Abbiamo isolato lo strato endoderma embrionale da YSM, e affettato la membrana in 2 - 3 pezzi mm e parzialmente digerito con collagenasi prima della semina in piastre di coltura da 24 pozzetti. Il TEE proliferano fuori del tessuto e sono pronti per studi su colture cellulari. Abbiamo trovato che il TEE aveva caratteristiche tipiche di YSM in vivo, per esempio, l&#39;accumulo di gocce lipidiche, espressione di steroli O-aciltransferasi e lipoproteina lipasi. Il trattamento di digestione parziale significativamente aumentato il tasso di successo della cultura CEE. Utilizzando il TEE, abbiamo dimostrato che l&#39;espressione di SOAT1 è stata regolata dal cAMP dla proteina chinasi A ependent percorso relativo. Questo sistema di coltura quaglia CEE giapponese primario è uno strumento utile per lo studio embrionale trasporto dei lipidi e di chiarire il ruolo dei geni coinvolti nella mediazione di utilizzo dei nutrienti nel YSM durante lo sviluppo embrionale aviaria.

Al fine di raggiungere l&#39;obiettivo di stabilire un modello cellulare coerente e utile, abbiamo bisogno di estendere e stabilizzare il tasso di proliferazione e le prestazioni dei TEE aviaria. Abbiamo confrontato incubazione diretta del endoderma senza digestione enzimatica con endoderma parzialmente digerito con gli enzimi proteolitici, come la collagenasi o collagenasi più 0,6 U di dispasi. Dispasi è un ammino-endopeptidasi che idrolizza i legami peptidici N-terminale di residui d...

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

Primaria endodermico cellule epiteliali Cultura dal tuorlo Sac membrana di giapponesi Quail embrioni

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

primary-endodermal-epithelial-cell-culture-from-yolk-sac-membrane

Research

JoVE Journal

Developmental Biology

8.8K Views.  National Taiwan University. To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

Video: Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

Trasferimento efficiente Gene in Chick retine per cellulari primarie Cultura Studies: An<em&gt; Ex-ovo</em&gt; Elettroporazione Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Ricerca

Biologia dello sviluppo

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolamento e caratterizzazione di singole cellule da embrioni di zebrafish

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Produzione su larga scala dei cardiomiociti da cellule staminali umane pluripotenti usando un altamente riproducibile piccole molecole-Based Differenziazione protocollo

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Affinità Etichettatura Rilevazione di endogena Recettori da embrioni di zebrafish

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Biosensing potenziale del membrane del neurone del motore in embrioni di Zebrafish in tensione

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

Registrazioni Patch-clamp intere cellule delle cellule epiteliali primarie isolate dall&#39;epididimo

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Derivazione di linee di cellule staminali da embrioni di Preimplantation Mouse

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

AFM e Microrheology nella cella tuorlo embrione di pesce zebra

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

Applicazione del sistema di coltura Explant Aorta-gonade-Mesonefro nell'ematopoiesi inerente allo sviluppo

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

L'isolamento e coltura di cellule primarie dell'epicardio ricavate da campioni di cuore umano adulto e fetale

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...