Les auteurs tiennent à remercier le financement fourni par le programme d&#39;allocation de ressources UCSF.

Tous les travaux des animaux a été effectuée sous stricte conformité avec les recommandations du Guide pour le soin et l&#39;utilisation des animaux de laboratoire des National Institutes of Health. Le protocole a été approuvé par le Comité institutionnel de protection des animaux et l&#39;utilisation (IACUC) de l&#39;Université de Californie, San Francisco. 1. souris embryonnaires glandes lacrymales (LG): récolte et microdissection <ol><li> Conformément aux procédures approuvées par les animaux institutionnels pour comité éthique de la recherche scientifique, euthanasier chronométré enceintes CD-1 (ou transgéniques) femelles avec la hausse inhalation de CO 2, suivie d&#39;une confirmation par dislocation cervicale aux embryons de récolte le jour embryonnaire approprié. Désigner le jour de prise vaginale découverte e0. Note: les glandes lacrymales (LGS) peut être récolté à partir de la E14 en une seule étape de bourgeon partir. Cependant, e16 (5 - 10 bourgeons) des embryons donnent les résultats les plus cohérents pour culture ex vivo. </li><li> Stériliser la ventral côté de la souris en utilisant 70% d&#39;éthanol. Pincer la peau et faire une incision sur la ligne médiane avec des ciseaux chirurgicaux stériles; couper à travers la peau et du péritoine pour exposer la cavité abdominale. Retirer les deux cornes utérines en coupant le long de la mésomètre au sommet de chaque corne utérine et dans du PBS froid (Phosphate Buffered Saline) ou du milieu DMEM F12 supplémenté avec 100 UI / ml de pénicilline et 100 ug / ml de streptomycine. </li><li> En utilisant des pinces (n ° 5), supprimer les embryons des sacs amniotiques et placer dans un deuxième boîte de Pétri stérile contenant du PBS froid. Attention à ne pas toucher les yeux ou la zone de la tête. </li><li> Passer embryon sur un microscope à dissection avec une base de transillumination. Si l&#39;embryon est &lt;e17, effectuez les étapes 1.5 à 1.7. Si les embryons sont&gt; e17, ces étapes peuvent être utiles mais ne sont pas indispensables, donc passez à l&#39;étape 1.8. </li><li> De trancher la tête du torse juste en dessous de la mandibule inférieure (étape 1, figure 2) usinga scalpel stérile (lame # 10). Tournez la tête de sorte que la mandibule est maintenant sur la plaque de dissection. Utilisez une pince à stabiliser la tête et un scalpel pour enlever environ ¼ de la face dorsale du cerveau (étape 2, 1 er coupe, Figure 2) - ce qui est particulièrement important une fois que le cartilage se raidit autour e15. </li><li> Orientez la tête pour que le scalpel peut percer le nez entre les yeux. Faire une coupe à travers le centre de la tête, de sorte que les yeux sont maintenant sur ​​moitiés séparées (étape 2, 2 e coupe, Figure 2). </li><li> L&#39;utilisation de deux # 5 forceps, prendre la moitié de la tête et enlever l&#39;excès de tissu (voir l&#39;étape 3, Figure 2). Veillez à orienter la tête de sorte que le LG (situé dans le coin inférieur arrière de l&#39;œil) ne soit pas perdu dans ce processus de retrait. Enlever l&#39;excès cérébrale, le cartilage et le tissu environnant nasale et sous l&#39;œil. Depuis la glande est à peine visible à ce stade, d&#39;assurer l&#39;oeil est bien orienté après l&#39;excès de tissu retrait to éviter de perdre l&#39;emplacement de la LG. </li><li> Prendre soin de la peau de la partie postérieure, coin inférieur de l&#39;oeil avec deux pinces. Retirer la peau en tirant avec précaution ouvert avec la pince pour exposer le LG. Utilisez un éclairage supplémentaire au-dessus et en dessous du tissu pour aider à visualiser le LG. Remarque: Le LG, distingue par son aspect comme un bourgeon sur un conduit dans un mésenchyme condensé sombre, doit être visible à ce stade. </li><li> Soigneusement commencent à disséquer le LG et le mésenchyme associé (voir illustration et images) à l&#39;écart du tissu environnant en utilisant une pince, en faisant attention de ne pas attraper le LG ou sa conduite associée. Une fois que la glande est libre du tissu environnant, retirez délicatement la totalité de la glande en saisissant l&#39;épithélium entourant l&#39;œil à la base du conduit LG. Prenez soin de préserver le mésenchyme entourant l&#39;épithélium, qui peut être observé comme un mésenchyme condensé dans lequel les invagine de l&#39;épithélium. NOTE: Certains élimination supplémentaire de tissue (des petits fragments d&#39;os, des muscles et du tissu conjonctif) peuvent être nécessaires pour éviter les facteurs / croissance signalisation de tissus voisins. </li><li> Pour la culture, la récolte 4 - 5 glandes et mésenchyme associé par plaque (GL sont déposées immédiatement sur dissection); recueillir un minimum de 14 glandes par 100 pi de tampon de lyse ARN pour RT-PCR 6. </li></ol> 2. Préparer les boîtes pour culture ex vivo Cette procédure est basée sur celle utilisée pour le développement des glandes salivaires 7,8. <ol><li> Ajouter 200 ul de milieu DMEM F12 (supplémenté avec 100 UI / ml de pénicilline et 100 ug / ml de streptomycine), des milieux de culture avec 50 ug / ml d&#39;acide ascorbique et de 50 pg / ml de holo-transferrine à une 50 mm plat à fond de verre de diamètre de microtitration 7. Remarque: DMEM F12 a été choisi comme nous l&#39;avons déjà constaté à maximiser la morphogenèse de la glande salivaire embryonnaire. </li><li> Flotter un 13 mm polycarbfiltre à membrane Onate avec 0,1 um pores sur le dessus de médias. </li><li> Étape facultative: Diluer 3-D laminine 1: 1 avec du DMEM / F12 pour obtenir une concentration finale de 3 mg / ml (utiliser 200 pi pipette pour mélanger doucement; centrifugeuse pendant 1 min si des bulles apparaissent). Ajouter 15 ul de cette dilution 3-D laminine à filtrer. Remarque: Cette dilution a été trouvée comme étant optimale pour le maintien à l&#39;état LG 3D sans compromettre la ramification épithéliale. Cependant, il est pas indispensable pour la culture de LG. </li><li> Placez 4 - 5 LG sur filtre ou dans la laminine. Culture GL ex vivo pour 24 - 48 h (figure 3) ou fixer avec 4% de PFA pendant 20 minutes pour une analyse plus approfondie par qPCR ou immunomarquage (Figure 4). Pour l&#39;analyse qPCR de tissu glandulaire embryonnaire, se il vous plaît se référer à Rebustini et al., 2011. Pour l&#39;analyse d&#39;immunofluorescence de tissus glandulaires embryonnaires se il vous plaît se référer aux citations suivantes: Hoffman et al, (2002) et Steinberg e.t al., (2005). </li></ol> 3. Souris postnatale et adultes glandes lacrymales (LG): Dissection <ol><li> Euthanasier la souris postnatale selon les normes de l&#39;éthique animale appropriée comité. Pour les petits postnatales jour 8 ou plus jeunes, euthanasier en coupant la tête avec des ciseaux stériles. Pour le jour postnatal 8 ou plus, pulvériser fourrure avec 70% d&#39;éthanol. </li><li> Pour localiser le LG, gisait souris latérale du côté sous un microscope à dissection avec un éclairage par le dessus. Remarque: Le LG post-natal et adulte sont bordés par l&#39;artère carotide, le muscle masséter et le derme (voir Figure 5). </li><li> L&#39;utilisation de petits ciseaux de dissection, faire petite incision dans l&#39;épiderme latéralement d&#39;où doit être situé le LG. </li><li> En utilisant des pinces, ouvrez le épiderme vers l&#39;oreille pour exposer la LG. </li><li> Utilisez une pince pour desserrer doucement LG du tissu environnant. </li><li> Une fois que le LG est libéré du tissu environnant, retirez délicatement le LG par le conduit,par pincement où le conduit et l&#39;épithélium entourant l&#39;oeil connecter (figure 1). </li><li> Placez GL dans l&#39;ARN tampon de lyse pour la RT-PCR, ou fixer avec 4% de PFA pendant 20 min pour immunomarquage. </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>

Les auteurs ont rien à divulguer.

La polyvalence de la culture et de manipuler le LG ex vivo apporte des avantages significatifs pour l&#39;étude de son développement. Cela inclut la vitesse à laquelle le chercheur est en mesure de tester les hypothèses et la multitude de perturbations qui peuvent être effectuées pour évaluer comment épithéliale, neuronale, les cellules endothéliales et mesencyhmal interagissent pour former l&#39;organe. Cependant, il ya un certain nombre de réserves lors de l&#39;utilisation de ce modèle. Tout d&#3...

La glande lacrymale (LG) est responsable de la sécrétion aqueuse déchirure critique pour l&#39;acuité visuelle et de la santé, la maintenance et la protection des cellules de la surface oculaire. Résultats de la dysfonction LG dans l&#39;un des troubles oculaires les plus fréquentes et invalidantes: Eye Disease aqueuse déficient à sec, qui se caractérise par une irritation oculaire, sensibilité à la lumière et diminution de la vision 1. Chez l&#39;homme le LG réside dans l&#39;orbite au-dessus de l&#39;extrémité latérale de l&#39;œil, où 3-5 excréteurs larmes de dépôt de conduits sur la surface oculaire. La souris possède trois paires de glandes majeures oculaires, le plus étudié de qui est la glande lacrymale (LG) situé antérieure et ventrale de l&#39;oreille (exorbital) avec des larmes qui voyagent à l&#39;œil par un seul canal excréteur. Semblable à d&#39;autres organes glandulaires, le LG développe à travers le processus de morphogenèse épithéliale ramification dans laquelle un seul bourgeon épithélial dans un mésenchyme condensé subit de multiples tours de bourgeon et la formation de conduit à desm un réseau interconnecté complexe d&#39;acini et canaux de sécrétion (Figure 1) 2. Au cours du développement de l&#39;épithélium se vascularise ainsi que fortement innervée par les nerfs parasympathique du ganglion ptérygo et dans une moindre mesure par les nerfs sympathiques du ganglion cervical supérieur 3. Les interactions entre les cellules de chacun de ces types à-dire les cellules neuronales, cellules épithéliales, endothéliales et mésenchymateuses, sont essentiels pour la fonction et la maintenance du tissu adulte. Cependant, les mécanismes moléculaires sous-jacents de coordination du développement et de la régénération LG ainsi que la façon de type inter-cellule communication guide ces processus reste incertaine. L&#39;avènement de la embryonnaires ex vivo des techniques de culture a permis l&#39;identification de voies de développement et de régénération dans plusieurs organes de branchement 4. La mise en culture ex vivo permet au chercheur la possibilité de manipuler l&#39;organe (moiméca-, génétique ou chimique) dans des conditions définies, ainsi que pour la caractérisation des interactions de développement d&#39;organes et cellule-cellule en temps réel. Le LG exorbital de la souris est prête très bien à cette technique et des études récentes ont défini les systèmes qui régissent son développement 2,5 signalisation. Cependant, en dépit de la nécessité de comprendre les signaux moléculaires qui sous-tendent le développement et la régénération LG, il reste actuellement étudié, probablement en raison des difficultés techniques pour isoler l&#39;organe. Dans cet article, nous décrivons comment isoler et effectuer culture ex vivo de l&#39;embryon murin LG pour définir des programmes de développement.

<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 glande lacrymale (LG) sécrète des larmes aqueuses nécessaires pour maintenir la structure et la fonction de la cornée, un tissu transparent essentielle pour la vision. Chez l&#39;être humain d&#39;une seule LG réside dans l&#39;orbite au-dessus de l&#39;extrémité latérale de chaque oeil délivrer larmes à la surface oculaire par l&#39;intermédiaire de 3 à 5 canaux. La souris possède trois paires de glandes majeures oculaires, le plus étudié de qui est la glande lacrymale exorbital (LG) situé antérieure et ventrale à l&#39;oreille. Semblable à d&#39;autres organes glandulaires, le LG développe à travers le processus de morphogenèse épithéliale ramification dans laquelle un seul bourgeon épithélial dans un mésenchyme condensé subit de multiples tours de bourgeon et la formation de conduite pour former un réseau interconnecté complexe de acini et canaux de sécrétion. Ce processus complexe a été bien documentée dans de nombreux autres organes épithéliaux tels que le pancréas et les glandes salivaires. Toutefois, le LG a été beaucoup moins explorée et les mécanismes de contrôle de la morphogenèse sont mal sousrésisté. Nous pensons que cette sous-représentation comme un système de modèle est une conséquence des difficultés liées à la recherche, la dissection et la culture de la LG. Ainsi, ici, nous décrivons les techniques de dissection pour la récolte embryonnaire et post-natal LG et les méthodes de culture ex vivo des tissus.

Le LG développe à travers le processus de morphogenèse épithéliale ramification. Images en fond clair de GL embryonnaires disséquées e14, e15, e16, e17, et P2 illustrent cet événement. <img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig1highres.jpg" width="600" /> Figure 1. Le LG développe à travers le processus de morphogenèse de ramification épithéliale. Images fond clair de GL embryonnai...

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Manipulating the Murine Lacrimal Gland

La glande lacrymale (LG) est un organe de branchement qui produit les composants aqueux de larmes nécessaires pour le maintien de la vision et la santé oculaire. Nous décrivons ici murin LG dissection et ex vivo des techniques de culture de déchiffrer les voies impliquées dans le développement LG signalisation.

Manipuler le murin Glande lacrymale

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 vues.  University of California San Francisco.  La glande lacrymale (LG) est un organe de branchement qui produit les composants aqueux de larmes nécessaires pour le maintien de la vision et la santé oculaire. Nous décrivons ici murin LG dissection et ex vivo des techniques de culture de déchiffrer les voies impliquées dans le développement LG signalisation. 

Vidéo: 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.

Les micropuces Silicon pour la manipulation d&#39;interaction cellule-cellule

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

Recherche

Biologie

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.

L&#39;aide de pincettes laser pour la manipulation neurones isolés 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.

Murin procédure transplantation rénale

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.

L&#39;isolement des cellules souches de la souris salivaires Gland

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.

Modification génétique et la recombinaison des cultures d&#39;organes salivaires Gland

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.

Injection intra-canalaire pour localisée Drug Delivery de la glande mammaire de souris

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.

Isolement des cellules endothéliales murines 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.

Isolement de murin coronaire vasculaires cellules de muscle lisse

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.

Techniques de biopsie des glandes mammaires bovine

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.

Isolement des cellules myoépithéliales des glandes murines lacrimal et sous-mandibulaires adultes

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, ...