Los autores desean agradecer el financiamiento proporcionado por el Programa de Asignación de Recursos de la UCSF.

Todo el trabajo de los animales se realizó en estricta conformidad con las recomendaciones de la Guía para el Cuidado y Uso de Animales de Laboratorio de los Institutos Nacionales de Salud. El protocolo fue aprobado por el Cuidado y Uso de Animales Comité Institucional (IACUC) de la Universidad de California, San Francisco. 1. embrionarias de ratón lagrimal Glándulas (LG): La recolección y Microdisección <ol><li> Siguiendo los procedimientos aprobados por los Animales institucionales para comité de ética de la investigación científica, la eutanasia programada embarazadas CD-1 (o transgénicos) las hembras con el aumento de inhalación de CO2, seguido de confirmación por dislocación cervical a los embriones de cosecha en el día embrionario adecuado. Designar el día de la vagina e0 descubrimiento enchufe. Nota: las glándulas lagrimales (LGS) puede ser cosechada desde el escenario solo brote e14 en adelante. Sin embargo, e16 (5-10 yemas) embriones dan los resultados más consistentes para el cultivo ex vivo. </li><li> Esterilizar la ventral lado del ratón usando etanol al 70%. Apriete la piel y hacer una incisión en la línea media con unas tijeras quirúrgicas estériles; cortar a través de la piel y el peritoneo para exponer la cavidad abdominal. Retire los 2 cuernos uterinos cortando a lo largo de la mesometrio en la parte superior de cada cuerno uterino y el lugar en PBS frío (tampón fosfato salino) o medio DMEM F12 suplementado con 100 U / ml de penicilina y 100 mg / ml de estreptomicina. </li><li> Con unas pinzas (# 5), eliminar los embriones de las bolsas amnióticas y colocar en una segunda placa de Petri estéril que contiene PBS frío. Tenga cuidado de no tocar los ojos o la zona de la cabeza. </li><li> Coloque embrión en un microscopio de disección con una base de transiluminación. Si el embrión es &lt;e17, realice los pasos 1.5 a 1.7. Si los embriones son&gt; e17, estos pasos pueden ser útiles, pero no son esenciales, por lo tanto, continúe con el paso 1.8. </li><li> Cortar la cabeza del torso justo por debajo de la mandíbula inferior (paso 1, Figura 2) usinga bisturí estéril (# 10 cuchilla). Girar la cabeza de manera que la mandíbula se encuentra ahora en la placa de disección. Utilice pinzas para estabilizar la cabeza y el bisturí para eliminar alrededor de ¼ de la parte dorsal del cerebro (el paso 2, 1 de corte, figura 2) - esto es especialmente importante una vez que el cartílago se endurece alrededor de e15. </li><li> Oriente la cabeza para que el bisturí puede perforar la nariz entre los ojos. Haga un corte a través del centro de la cabeza, de modo que los ojos están puestos en mitades separadas (paso 2, 2º corte, Figura 2). </li><li> El uso de dos # 5 pinzas, tomar la mitad de la cabeza y quite el exceso de tejido (véase el paso 3, Figura 2). Asegúrese de orientar la cabeza por lo que el LG (que se encuentra en la esquina posterior inferior del ojo) no se pierde en este proceso de eliminación. Eliminar el exceso de cerebral, cartílago y tejido nasal circundante y debajo del ojo. Puesto que la glándula es apenas visible en este momento, garantizar el ojo está orientada correctamente después de exceso de tejido eliminación to evitar la pérdida de la ubicación de la LG. </li><li> Agarrar con cuidado la piel de la parte posterior, en la esquina inferior del ojo con las dos pinzas. Quitar la piel tirando cuidadosamente abierta con las pinzas para exponer el LG. Utilice iluminación adicional por encima y por debajo del tejido para ayudar a visualizar el LG. Nota: El LG, distinguibles por su aparición como un botón en un conducto dentro de un mesénquima condensado más oscuro, debe ser visible en este punto. </li><li> Empezar con cuidado para diseccionar el LG y mesénquima asociado (consulte el diagrama e imágenes) de distancia del tejido que rodea el uso de fórceps, teniendo cuidado de no agarrar el LG o su conducto asociado. Una vez que la glándula está libre del tejido circundante, eliminar suavemente toda la glándula agarrando el epitelio que rodea el ojo en la base del conducto de LG. Tenga cuidado para preservar el mesénquima que rodea el epitelio, que se puede observar como un mesénquima condensado en el que los invagina epitelio. NOTA: Algunos eliminación adicional de tissue (los pequeños fragmentos de hueso, músculo y tejido conectivo) pueden ser necesarias para evitar factores de señalización / crecimiento de los tejidos vecinos. </li><li> Para el cultivo, la cosecha 4 - 5 glándulas y mesénquima asociado por placa (GL se colocan inmediatamente después de la disección); recoger un mínimo de 14 ganglios por cada 100 l de tampón de lisis de ARN por RT-PCR 6. </li></ol> 2. Prepare las placas para ex vivo Cultura Este procedimiento se basa en que la empleada para el desarrollo de la glándula salival 7,8. <ol><li> Añadir 200 l de DMEM F12 (suplementado con 100 U / ml de penicilina y 100 mg / ml de estreptomicina) medios de cultivo con 50 mg / ml de ácido ascórbico y 50 mg / ml holo-transferrina a un plato de 50 mm de diámetro de micropocillos con fondo de vidrio 7. Nota: F12 DMEM fue elegido ya que previamente nos pareció que para maximizar la morfogénesis de la glándula salival embrionaria. </li><li> Flotador un Polycarb 13 mmfiltro de membrana de 0,1 micras Oñate con poros en la parte superior de los medios de comunicación. </li><li> Paso opcional: Diluir laminina 3-D 1: 1 con DMEM / F12 para hacer una concentración final de 3 mg / ml (200 l utilizar la pipeta para mezclar suavemente; centrifugar durante 1 min si aparecen burbujas). Añadir 15 l de esta diluyeron laminina 3-D para filtrar. Nota: Esta dilución se encontró que era óptimo para el mantenimiento de la LG en su estado 3D sin comprometer ramificación epitelial. Sin embargo, no es esencial para la cultura de LG. </li><li> Coloque 4 - 5 LG en filtro o en laminina. Cultura GL ex vivo por 24 - 48 h (Figura 3) o fijar con PFA al 4% durante 20 min para su posterior análisis por qPCR o inmunotinción (Figura 4). Para el análisis qPCR de tejido glandular embrionario, por favor consulte Rebustini et al., 2011. Para el análisis de inmunofluorescencia de los tejidos glandulares embrionarias por favor consulte las siguientes citas: Hoffman et al, (2002) y Steinberg correo.t al., (2005). </li></ol> 3. Ratón postnatal y adulto lagrimal Glándulas (LG): Disección <ol><li> La eutanasia del ratón postnatal de acuerdo a las normas del comité de ética animal apropiado. Para cachorros postnatal día 8 o más jóvenes, la eutanasia por el corte de la cabeza con unas tijeras estériles. Para el día postnatal 8 años o más, rociar la piel con etanol al 70%. </li><li> Para localizar el LG, yacía ratón lateral lado bajo un microscopio de disección con iluminación desde arriba. Nota: El LG postnatal y adulto se limita con la arteria carótida, el músculo masetero y la dermis (ver Figura 5). </li><li> El uso de pequeñas tijeras de disección, hacer pequeña incisión en la epidermis lateralmente desde donde se debe colocar el LG. </li><li> Con unas pinzas, abra la epidermis hacia la oreja para exponer el LG. </li><li> Utilice pinzas para aflojar suavemente LG del tejido circundante. </li><li> Una vez que el LG se libera del tejido circundante, retire con cuidado el LG por el conducto,agarrando donde el conducto y el epitelio que rodea el ojo se conectan (Figura 1). </li><li> Coloque los GL en tampón de lisis de ARN para RT-PCR, o fijar con PFA al 4% durante 20 min para inmunotinción. </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>

Los autores no tienen nada que revelar.

La versatilidad de la cultura y manipular el LG ex vivo ofrece ventajas significativas para el estudio de su desarrollo. Esto incluye la velocidad a la cual el investigador es capaz de probar las hipótesis y la multitud de las perturbaciones que se pueden realizar para evaluar cómo epitelial, neuronal, endotelial y células mesencyhmal interactúan para formar el órgano. Sin embargo, hay una serie de advertencias cuando se utiliza este modelo. En primer lugar, en virtud de su aislamiento de la glándula ya n...

La glándula lagrimal (LG) es responsable de la secreción de lágrima acuosa crítica para la agudeza visual y la salud, el mantenimiento y la protección de las células de la superficie ocular. LG resultados disfunción en uno de los trastornos oculares más comunes y debilitantes: acuoso deficiente en seco de Enfermedades de los ojos, que se caracteriza por la irritación ocular, sensibilidad a la luz y la disminución de la visión 1. En el ser humano la LG reside en la órbita por encima del extremo lateral del ojo donde 3 - 5 excretores lágrimas de depósito conductos sobre la superficie ocular. El ratón tiene tres pares de glándulas oculares, el más estudiado de los cuales es la glándula lagrimal (LG) situado anterior y ventral de la oreja (exorbital) con lágrimas que viajan al ojo a través de un solo conducto excretor. Similar a otros órganos glandulares, el LG desarrolla a través del proceso de morfogénesis epitelial de ramificación en el que un solo brote epitelial dentro de un mesénquima condensado se somete a múltiples rondas de brote y la formación del conducto a parasoy una intrincada red interconectada de acinos y conductos de secreción (Figura 1) 2. Durante el desarrollo del epitelio se vasculariza, así como en gran medida inervado por los nervios parasimpáticos del ganglio pterigopalatina y en menor medida por los nervios simpáticos del ganglio cervical superior 3. Las interacciones entre cada uno de estos tipos de células es decir, células neuronales, epiteliales, endoteliales y mesenquimales, son esenciales para la función y el mantenimiento del tejido adulto. Sin embargo, los mecanismos moleculares subyacentes de coordinación de desarrollo LG y regeneración, así como la forma en guías de comunicación de tipo de células entre estos procesos sigue siendo poco clara. El advenimiento de las embrionarias ex vivo las técnicas de cultivo ha permitido la identificación de las vías de desarrollo y regenerativos en múltiples órganos de ramificación 4. El cultivo ex vivo da el investigador la capacidad de manipular el órgano (memecánico, genético o químico) bajo condiciones definidas, así como para caracterizar el desarrollo de órganos y las interacciones célula-célula en tiempo real. El LG exorbital del ratón es altamente susceptible a esta técnica y estudios recientes han definido sistemas que regulan su desarrollo 2,5 señalización. Sin embargo, a pesar de la necesidad de comprender las señales moleculares que sustentan el desarrollo de LG y la regeneración, en la actualidad sigue siendo poco estudiada, probablemente debido a las dificultades técnicas para aislar el órgano. En este trabajo se describe cómo aislar y llevar a cabo el cultivo ex vivo de la embrionaria murina LG para definir los programas de desarrollo.

<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 glándula lagrimal (LG) segrega lágrimas acuosas necesarias para mantener la estructura y la función de la córnea, un tejido transparente esencial para la visión. En el ser humano una sola LG reside en la órbita sobre el extremo lateral de cada ojo la entrega de lágrimas a la superficie ocular a través de 3 - 5 conductos. El ratón tiene tres pares de glándulas oculares, el más estudiado de los cuales es la glándula lagrimal exorbital (LG) situado anterior y ventral de la oreja. Similar a otros órganos glandulares, el LG desarrolla a través del proceso de morfogénesis epitelial de ramificación en el que un solo brote epitelial dentro de un mesénquima condensado se somete a múltiples rondas de brote y la formación del conducto para formar una red interconectada intrincada de los acinos y conductos de secreción. Este elaborado proceso ha sido bien documentado en muchos otros órganos epiteliales, tales como el páncreas y la glándula salival. Sin embargo, el LG ha sido mucho menos explorada y los mecanismos que controlan la morfogénesis son mal bajoresistido. Tenemos la sospecha de que esta falta de representación como un sistema modelo es una consecuencia de las dificultades relacionadas con la búsqueda, la disección y el cultivo de la LG. Por lo tanto, aquí se describen técnicas de disección de embriones cosecha y post-natal LG y métodos de cultivo ex vivo de los tejidos.

El LG desarrolla a través del proceso de la morfogénesis de ramificación epitelial. Imágenes de campo claro de los gobiernos locales de embriones disecados en e14, e15, e16, e17, y P2 ilustran este evento. <img alt="Figura 1" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig1highres.jpg" width="600" /> Figura 1. El LG desarrolla a través del proceso de morfogénesis de ramificación epitelial. Imágenes de campo clar...

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Manipulating the Murine Lacrimal Gland

La glándula lagrimal (LG) es un órgano de ramificación que produce los componentes acuosos de lágrimas necesarias para el mantenimiento de la visión y la salud ocular. Aquí describimos murino LG disección y ex vivo las técnicas de cultivo de descifrar las vías de señalización implicadas en el desarrollo de LG.

La manipulación de la murino Glándula Lagrimal

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 vistas.  University of California San Francisco.  La glándula lagrimal (LG) es un órgano de ramificación que produce los componentes acuosos de lágrimas necesarias para el mantenimiento de la visión y la salud ocular. Aquí describimos murino LG disección y ex vivo las técnicas de cultivo de descifrar las vías de señalización implicadas en el desarrollo de 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.

Los microchips de silicio para la Manipulación de la interacción célula-célula

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

Investigación

Biología

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.

Utilizando pinzas láser para manipular neuronas aisladas 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.

Procedimiento murino Trasplante Renal

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.

Aislamiento de células de la glándula salival del ratón madre

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.

Modificación genética y recombinación de las Glándulas Salivales culturas de órganos

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.

Inyección intraductal de entrega de la droga localizada a la glándula mamaria de ratón

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.

Aislamiento de las células murinas de la válvula endoteliales

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

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Inmunofluorescencia indirecta en secciones congeladas de ratón Glándula Mamaria

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

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.

Aislamiento de murino coronaria células musculares lisas vasculares

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.