Gli autori desiderano ringraziare finanziamenti previsti dal Programma di allocazione delle risorse UCSF.

Tutto il lavoro degli animali sono state eseguite in stretta conformità con le raccomandazioni nella Guida per la cura e l&#39;uso di animali da laboratorio del National Institutes of Health. Il protocollo è stato approvato dalla cura e l&#39;uso Comitato Istituzionale Animal (IACUC) della University of California, San Francisco. 1. embrionali di topo lacrimale Ghiandole (LG): Raccolta e Microdissection <ol><li> A seguito di procedure approvate dalle Animali istituzionali per comitato della ricerca scientifica etica, eutanasia cronometrato in gravidanza CD-1 (o transgenici) femmine con l&#39;aumento della CO 2 inalazione, seguita da una conferma per dislocazione cervicale di embrioni consecutivi nella appropriata giorno embrionale. Designare il giorno della scoperta vaginale spina e0. Nota: ghiandole lacrimali (LGS) può essere raccolto dalla fase singola gemma E14 in poi. Tuttavia, E16 (5-10 gemme) embrioni danno i risultati più coerenti per ex vivo della cultura. </li><li> Sterilizzare il vlato entral del mouse utilizzando etanolo al 70%. Pizzicare la pelle e fare un&#39;incisione sulla linea mediana con le forbici chirurgiche sterili; tagliare attraverso la pelle e del peritoneo per esporre la cavità addominale. Rimuovere le 2 corna uterine tagliando lungo il mesometrio nella parte superiore di ogni corno uterino e posto in PBS freddo (Phosphate Buffered Saline) o DMEM F12 supporto integrato con 100 U / ml di penicillina e 100 ug / ml di streptomicina. </li><li> Utilizzando pinze (# 5), rimuovere gli embrioni dalle cavità amniotiche e mettere in una seconda piastra di Petri sterile contenente PBS freddo. Fare attenzione a non toccare gli occhi o la zona della testa. </li><li> Inserire embrione su un microscopio da dissezione con una base transilluminazione. Se l&#39;embrione è &lt;E17, eseguire i passaggi 1,5-1,7. Se gli embrioni sono&gt; E17, questa procedura può essere utile, ma non sono essenziali, quindi procedere al passaggio 1.8. </li><li> Sever testa dal tronco appena sotto la mandibola inferiore (fase 1, Figura 2) Struga bisturi sterile (# 10 lama). Ruotare testa in modo che la mandibola è ora su piastra dissezione. Utilizzare pinze per stabilizzare la testa e un bisturi per rimuovere circa ¼ del lato dorsale del cervello (fase 2, 1 ° taglio, Figura 2) - questo è particolarmente importante una volta che la cartilagine si irrigidisce intorno e15. </li><li> Orientare la testa in modo che il bisturi può perforare il naso tra gli occhi. Effettuare un taglio attraverso il centro della testa, in modo che gli occhi sono ora a metà separate (step 2, 2 ° taglio, Figura 2). </li><li> Utilizzando due # 5 pinze, prendere una metà della testa e rimuovere il tessuto in eccesso (vedere il punto 3, figura 2). Accertarsi di orientare la testa in modo che il LG (che si trova nell&#39;angolo posteriore inferiore dell&#39;occhio) non si perde in questo processo di rimozione. Rimuovere cerebrale in eccesso, cartilagine e tessuto nasale circostante e sotto l&#39;occhio. Dal momento che la ghiandola è appena visibile, a questo punto, assicurare l&#39;occhio sia correttamente orientata tessuto in eccesso dopo la rimozione to evitare di perdere la posizione del LG. </li><li> Afferrare con cura la pelle dalla parte posteriore, in basso dell&#39;occhio con entrambe le pinze. Togliere la pelle tirando delicatamente aperto con le pinze per esporre la LG. Utilizzare l&#39;illuminazione supplementare sopra e sotto il tessuto per aiutare a visualizzare la LG. Nota: L&#39;LG, distinguibile dal suo aspetto come gemma su un condotto all&#39;interno di una più scura, mesenchima condensato, deve essere visibile in questo punto. </li><li> Cominciare con cura per sezionare la LG e mesenchima associati (vedere lo schema e immagini) dal tessuto circostante usando pinze, facendo attenzione a non afferrare il LG o il suo canale associato. Una volta che la ghiandola è libero dal tessuto circostante, rimuovere delicatamente l&#39;intera ghiandola afferrando la epiteli circonda l&#39;occhio alla base del condotto LG. Fare attenzione a preservare il mesenchima che circonda l&#39;epitelio, che può essere osservata come un mesenchima condensata in cui le invaginates epitelio. NOTA: Alcuni eliminata anche la tissue (eventuali piccoli frammenti di ossa, muscoli e tessuto connettivo) può essere necessario per evitare la segnalazione fattori / crescita dal tessuto adiacente. </li><li> Per la cultura, il raccolto 4 - 5 ghiandole e mesenchima associati per piastra (enti locali sono placcati immediatamente dopo la dissezione); raccogliere un minimo di 14 ghiandole per 100 ml di RNA tampone di lisi per RT-PCR 6. </li></ol> 2. Preparare Piastre per ex vivo Cultura Questa procedura si basa su quello utilizzato per lo sviluppo delle ghiandole salivari 7,8. <ol><li> Aggiungere 200 ml di DMEM F12 (integrato con 100 U / ml di penicillina e 100 mg / ml di streptomicina) terreni di coltura con 50 mg / ml di acido ascorbico e 50 mg / ml di olo-transferrina ad un diametro di 50 millimetri piatto micropozzetti con fondo trasparente 7. Nota: DMEM F12 è stato scelto come abbiamo precedentemente trovato per massimizzare morfogenesi nella ghiandola salivare embrionale. </li><li> Float un Polycarb 13 millimetrifiltro a membrana Onate con 0,1 micron pori sulla parte superiore del supporto. </li><li> Passaggio opzionale: Diluire 3-D laminina 1: 1 con DMEM / F12 per effettuare una concentrazione finale di 3 mg / ml (200 ml utilizzare pipetta per mescolare delicatamente; centrifugare per 1 minuto se compaiono bolle). Aggiungere 15 ml di questo diluiti 3-D laminina per filtrare. Nota: Questa diluizione è risultata ottimale per mantenere LG nel suo stato 3D senza compromettere ramificazione epiteliale. Tuttavia, non è essenziale per la cultura di LG. </li><li> Mettere 4-5 LG sul filtro o in laminina. Cultura LGs ex vivo per 24 - 48 ore (Figura 3) o fissare con PFA 4% per 20 min per ulteriori analisi qPCR o immunocolorazione (Figura 4). Per l&#39;analisi qPCR di tessuto ghiandolare embrionale, si prega di fare riferimento al Rebustini et al., 2011. Per l&#39;analisi di immunofluorescenza di tessuti ghiandolari embrionali si rimanda ai seguenti citazioni: Hoffman et al, (2002) e Steinberg e.t al., (2005). </li></ol> 3. mouse postnatale e adulti ghiandole lacrimale (LG): Dissection <ol><li> Euthanize il mouse postnatale secondo gli standard del caso della commissione etica animale. Per cuccioli postnatale giorno 8 o più giovani, eutanasia tagliando la testa con le forbici sterili. Per giorno postnatale 8 o più anziani, spruzzare pelliccia con il 70% di etanolo. </li><li> Per individuare il LG, giaceva il mouse laterale all&#39;altro sotto un microscopio da dissezione con illuminazione dall&#39;alto. Nota: L&#39;LG post-natale e adulti sono confina con l&#39;arteria carotide, il muscolo massetere e il derma (vedi Figura 5). </li><li> Utilizzando piccole forbici dissezione, fare piccola incisione nell&#39;epidermide lateralmente da cui deve essere posizionato il LG. </li><li> Utilizzando pinze, aprire l&#39;epidermide verso l&#39;orecchio per esporre la LG. </li><li> Utilizzare pinze per allentare delicatamente LG dal tessuto circostante. </li><li> Una volta che la LG è liberato dal tessuto circostante, rimuovere con attenzione l&#39;LG dal condotto,afferrando dove il condotto e la epiteli circonda l&#39;occhio collegano (Figura 1). </li><li> Mettere enti locali in RNA tampone di lisi per RT-PCR, o fissare con 4% PFA per 20 minuti per immunocolorazione. </li></ol>

<ol>
	<li>Liu, K. C., Huynh, K., Grubbs, J., Davis, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+the+pathogenesis+and+treatment+of+keratoconjunctivitis+sicca.">Autoimmunity in the pathogenesis and treatment of keratoconjunctivitis sicca.</a> Current allergy and asthma reports. 14, 403 (2014).</li><li>Makarenkova, H. P., 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=FGF10+is+an+inducer+and+Pax6+a+competence+factor+for+lacrimal+gland+development.">FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.</a> Development. 127, 2563-2572 (2000).</li><li>Dartt, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regulation+of+lacrimal+gland+secretory+processes:+relevance+in+dry+eye+diseases.">Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases.</a> Progress in retinal and eye research. 28, 155-177 (2009).</li><li>Nigam, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+can+the+intrinsic+power+of+branching+morphogenesis+be+used+for+engineering+epithelial+tissues+and+organs.">Concise review: can the intrinsic power of branching morphogenesis be used for engineering epithelial tissues and organs.</a> Stem cells translational medicine. 2, 993-1000 (2013).</li><li>Qu, X., 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=Glycosaminoglycan-dependent+restriction+of+FGF+diffusion+is+necessary+for+lacrimal+gland+development.">Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development.</a> Development. 139, 2730-2739 (2012).</li><li>Rebustini, I. T., 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=MT2-MMP-dependent+release+of+collagen+IV+NC1+domains+regulates+submandibular+gland+branching+morphogenesis.">MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis.</a> Developmental cell. 17, 482-493 (2009).</li><li>Hoffman, M. P., 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=Gene+Expression+Profiles+of+Mouse+Submandibular+Gland+Development:+FGFR1+Regulates+Branching+Morphogenesis+in+Vitro+Through+BMP-+and+FGF-Dependent+Mechanisms.">Gene Expression Profiles of Mouse Submandibular Gland Development: FGFR1 Regulates Branching Morphogenesis in Vitro Through BMP- and FGF-Dependent Mechanisms.</a> Development. 129, 24-5778 (2002).</li><li>Steinberg, Z., 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=FGFR2b+signaling+regulates+ex+vivo+submandibular+gland+epithelial+cell+proliferation+and+branching+morphogenesis.">FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.</a> Development. 132, 1223-1234 (2005).</li><li>Knox, S. M., 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=.">.</a> Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. 329, 1645-1647 (2010).</li><li>Magenheim, J., 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=Blood+vessels+restrain+pancreas+branching,+differentiation+and+growth.">Blood vessels restrain pancreas branching, differentiation and growth.</a> Development. 138, 4743-4752 (2011).</li><li>Jaskoll, T., 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=FGF10/FGFR2b+signaling+plays+essential+roles+during+in+vivo+embryonic+submandibular+salivary+gland+morphogenesis.">FGF10/FGFR2b signaling plays essential roles during in vivo embryonic submandibular salivary gland morphogenesis.</a> BMC developmental biology. 5, 11 (2005).</li><li>Takasato, M., 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=Directing+human+embryonic+stem+cell+differentiation+towards+a+renal+lineage+generates+a+self-organizing+kidney.">Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.</a> Nature cell biology. 16, 118-126 (2014).</li><li>Knox, S. M., 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=Parasympathetic+stimulation+improves+epithelial+organ+regeneration.">Parasympathetic stimulation improves epithelial organ regeneration.</a> Nature communications. 4, 1494 (2013).</li></ol>

Gli autori non hanno nulla da rivelare.

La versatilità alla cultura e manipolare la LG ex vivo fornisce vantaggi significativi per lo studio del suo sviluppo. Ciò comprende la velocità alla quale il ricercatore è in grado di testare ipotesi e la moltitudine di perturbazioni che possono essere eseguite per valutare come epiteliale, neuronali, cellule endoteliali e mesencyhmal interagiscono per formare l&#39;organo. Tuttavia, ci sono una serie di avvertimenti quando si utilizza questo modello. In primo luogo, in virtù del suo isolamento ghiandola ...

La ghiandola lacrimale (LG) è responsabile della secrezione lacrimale acquoso critico per l&#39;acuità visiva e la salute, la manutenzione e la protezione delle cellule della superficie oculare. LG risultati disfunzione in uno dei disturbi oculari più comuni e debilitanti: acquosa carente malattia dell&#39;occhio secco, che è caratterizzata da irritazione oculare, sensibilità alla luce e riduzione della visione 1. Nell&#39;essere umano LG risiede nell&#39;orbita sopra dell&#39;estremità laterale dell&#39;occhio dove che 3 - 5 escrezione lacrime deposito condotti sulla superficie oculare. Il mouse ha tre paia di ghiandole oculari, il più studiato dei quali è la ghiandola lacrimale (LG) anterior trova e ventrale per l&#39;orecchio (exorbital) con le lacrime agli occhi che viaggiano attraverso un unico condotto escretore. Simile ad altri organi ghiandolari, LG si sviluppa attraverso il processo di morfogenesi epiteliale ramificazione in cui un singolo gemma epiteliale all&#39;interno di un mesenchima condensato sottoposto a vari cicli di gemme e la formazione del condotto di perm una rete interconnessa intricata di acini secretoria e condotti (Figura 1) 2. Durante lo sviluppo l&#39;epitelio viene vascolarizzato e fortemente innervato dai nervi parasimpatici del ganglio pterigopalatino e, in misura minore, da nervi simpatici del ganglio cervicale superiore 3. Interazioni tra ciascuno di questi tipi di cellule cioè cellule neuronali, epiteliali, endoteliali e mesenchimali, sono essenziali per la funzione e la manutenzione del tessuto adulto. Tuttavia, i meccanismi molecolari alla base di coordinamento sviluppo LG e la rigenerazione e come tipo di comunicazione inter-cellulare guida questi processi rimane poco chiaro. L&#39;avvento della embrionali ex vivo le tecniche di coltura ha consentito l&#39;individuazione di percorsi di sviluppo e di rigenerazione in più organi di derivazione 4. Coltura ex vivo dà al ricercatore la possibilità di manipolare l&#39;organo (memeccanico, genetica o chimica) in condizioni definite, nonché per caratterizzare le interazioni sviluppo organo e cellula-cellula in tempo reale. La exorbital LG del mouse è altamente suscettibile di questa tecnica e studi recenti hanno definito i sistemi che regolano il suo sviluppo 2,5 segnalazione. Tuttavia, nonostante la necessità di comprendere segnali molecolari alla base dello sviluppo LG e la rigenerazione, attualmente rimane poco studiato, probabilmente a causa delle difficoltà tecniche di isolare l&#39;organo. In questo articolo, si descrive come isolare ed eseguire ex vivo cultura del embrionale murino LG per definire programmi di sviluppo.

<table border="0" cellpadding="0" cellspacing="0" width="1767">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Name</td>
			<td style="width:129px;">Catalog #.</td>
			<td style="width:213px;">Company</td>
			<td style="width:997px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 without HEPES</td>
			<td>SH3027101</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>P0781</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin solution from bovine serum</td>
			<td>A9576</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% Solution</td>
			<td>15710</td>
			<td>Electron Microscopy Sciences</td>
			<td>Diluted to 4% in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 10X</td>
			<td>BP665-1</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 1X</td>
			<td></td>
			<td></td>
			<td>Prepared from 10X stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">holo-Transferrin bovine</td>
			<td>T1283</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">L- Ascorbic acid (Vitamin C)</td>
			<td>A4544</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite 100mm Tissue Culture Treated Dishes</td>
			<td>130182</td>
			<td>Thermo Scientific</td>
			<td>Non-tissue culture-treated plates can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo Microscope with substage iluminator</td>
			<td>Stemi 2000</td>
			<td>Carl Zeiss</td>
			<td rowspan="2">Any stereo dissecting microscope can be used that has a transmitted light base.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substage Illuminator Base for Stereo Microscope</td>
			<td>TLB 4000</td>
			<td>Diagnostics Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 35mm Tissue Culture Treated Dishes</td>
			<td>353001</td>
			<td>Corning</td>
			<td>Non-tissue culture-treated plates can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mm uncoated glass bottom dishes</td>
			<td>P50G-1.5-14-F</td>
			<td>MatTek Corporation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Widefield fluorescence microscope</td>
			<td>Axio Observer Z1</td>
			<td style="width:213px;">Carl Zeiss</td>
			<td>Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached 
			digital camera.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>TCS SP5</td>
			<td style="width:213px;">Leica Microsystems</td>
			<td>Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SWISS Micro-Fine Forceps, #5 (11.2cm)</td>
			<td>17-305X</td>
			<td>Integra LifeSciences Corporation</td>
			<td>Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Standard Tip Forceps, #5 (11cm)</td>
			<td>91150-20</td>
			<td>Fine Science Tools (USA), Inc.</td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reusable Plastic Surgical Knife Handles, style no. 3.</td>
			<td>4-30</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Sterile Surgical Blades, no. 10</td>
			<td>4-310</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAqueous-Micro Total RNA Isolation Kit</td>
			<td>AM1931</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cultrex 3D Culture Matrix Laminin I</td>
			<td>3446-005-01</td>
			<td>Trevigen</td>
			<td>6 mg/ml stock diluted 1:1 with DMEM/F12</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Timed-pregnant Crl:CD1(ICR) mice</td>
			<td></td>
			<td style="width:213px;">Charles River Labs</td>
			<td>Embryos are harvested on day 14 (with day of plug discovery designated as day 0).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nucleopore Track-Etched Hydrophilic Membranes, 0.1 um pre size, 13mm</td>
			<td>110405</td>
			<td>Whatman</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Timed-pregnant Pax6-Cre,GFP mice</td>
			<td>Tg(Pax-Cre, GFP)1Pgr</td>
			<td>The Jackson Laboratory</td>
			<td>Optional mice for learning how to locate the LG.</td>
		</tr>
	</tbody>
</table>

manipulating the murine lacrimal gland

La ghiandola lacrimale (LG) secerne lacrime acquose necessari a mantenere la struttura e la funzione della cornea, un tessuto trasparente essenziale per la visione. Nell&#39;essere umano un&#39;unica LG risiede nella orbita sopra l&#39;estremità laterale di ciascun occhio fornire lacrime alla superficie oculare attraverso 3 - 5 condotti. Il mouse ha tre paia di ghiandole oculari, il più studiato dei quali è la ghiandola lacrimale exorbital (LG) anterior posizionato e ventrale all&#39;orecchio. Simile ad altri organi ghiandolari, LG si sviluppa attraverso il processo di morfogenesi epiteliale ramificazione in cui un singolo gemma epiteliale all&#39;interno di un mesenchima condensato sottoposto a vari cicli di gemme e di formazione condotto da formare una rete interconnessa intricata di acini secretori e condotti. Questo processo elaborato è stato ben documentato in molti altri organi epiteliali come il pancreas e ghiandole salivari. Tuttavia, la LG è stata molto meno esplorati e meccanismi che controllano la morfogenesi sono poco sottoc&#39;era. Abbiamo il sospetto che questo sotto-rappresentazione come sistema modello è una conseguenza delle difficoltà connesse con l&#39;individuazione, la dissezione e la coltura della LG. Così, qui descriviamo tecniche di dissezione per la raccolta embrionale e post-natale LG e metodi di coltura ex vivo del tessuto.

La LG si sviluppa attraverso il processo di morfogenesi epiteliale ramificazione. Immagini in campo chiaro di enti locali embrionali sezionati a E14, E15, E16, E17, e P2 illustrano questo evento. <img alt="Figura 1" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig1highres.jpg" width="600" /> Figura 1. L&#39;LG si sviluppa attraverso il processo di morfogenesi epiteliale ramificazione. Immagini in campo chiaro di enti loc...

Watch this Scientific Journal Video about Manipulating the Murine Lacrimal Gland at JoVE.com

Manipulating the Murine Lacrimal Gland

La ghiandola lacrimale (LG) è un organo di ramificazione che produce i componenti acquose di lacrime necessarie per il mantenimento della visione e la salute oculare. Qui si descrive murino LG dissezione ed ex vivo le tecniche di coltura di decifrare vie di segnalazione coinvolte nello sviluppo di LG.

Manipolare il murino Ghiandola lacrimale

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Research

JoVE Journal

Biology

20.7K Views.  University of California San Francisco.  La ghiandola lacrimale (LG) è un organo di ramificazione che produce i componenti acquose di lacrime necessarie per il mantenimento della visione e la salute oculare. Qui si descrive murino LG dissezione ed ex vivo le tecniche di coltura di decifrare vie di segnalazione coinvolte nello sviluppo di LG. 

Video: Manipulating the Murine Lacrimal Gland

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

I microchip di silicio per la manipolazione di interazione cellula-cellula

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Ricerca

Biologia

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

Utilizzando pinzette laser per manipolare i neuroni isolato in vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murino procedura trapianto renale

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolamento di cellule staminali del mouse ghiandole salivari

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Modificazione genetica e ricombinazione delle Culture organo ghiandola salivare

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Iniezione intraduttale per Localizzato Drug Delivery per il mouse ghiandola mammaria

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolamento di cellule murine endoteliali Valve

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

L&#39;isolamento di Murine coronarica vascolari cellule muscolari lisce

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

Tecniche di biopsia della ghiandola mammaria bovina

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

Isolamento delle cellule Miepiteliali da adulti murina lacrimale e ghiandole sottomandibolari

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...