Os autores gostariam de agradecer financiamento concedido pelo Programa de Alocação de Recursos UCSF.

Todo o trabalho animal foi realizada sob estrita conformidade com as recomendações do Guia para o Cuidado e Uso de Animais de Laboratório do National Institutes of Health. O protocolo foi aprovado pelo cuidado e uso Comitê Institucional Animal (IACUC) da Universidade da Califórnia, em San Francisco. 1. embrionárias de camundongos lacrimais Glândulas (LG): Colheita e Microdissection <ol><li> Seguindo os procedimentos aprovados pelos Animais institucionais para a comissão de Ética de Pesquisa Científica, eutanásia cronometrado CD-1 (ou transgênicos) fêmeas grávidas com o aumento da inalação de CO 2, seguido de confirmação por deslocamento cervical para colheita de embriões no dia embrionário adequado. Designar o dia da vaginal e0 ficha descoberta. Nota: glândulas lacrimais (LGS) podem ser colhidas a partir da e14 único broto fase em diante. No entanto, e16 (5-10 gomos) embriões dar os resultados mais consistentes para ex vivo da cultura. </li><li> Esterilizar a vlado entral do mouse usando etanol 70%. Aperte a pele e fazer uma incisão na linha média com tesouras cirúrgicas estéreis; cortar através da pele e do peritoneu para expor a cavidade abdominal. Retirar as duas trompas uterinas por corte ao longo da mesométrio no topo de cada trompa uterina e em lugar frio PBS (Tampão fosfato salino) ou meio DMEM F12 suplementado com 100 U / ml de penicilina e 100 ug / ml de estreptomicina. </li><li> Utilizando uma pinça (# 5), remova os embriões dos sacos amniótico e coloque em uma segunda placa de Petri estéril contendo PBS frio. Tenha cuidado para não tocar os olhos ou a área da cabeça. </li><li> Coloque embrião em um microscópio de dissecação com uma base de transiluminação. Se o embrião é &lt;e17, execute os passos 1,5-1,7. Se os embriões são&gt; e17, estes passos podem ser úteis, mas não são essenciais, portanto, siga para o passo 1.8. </li><li> Cortar a cabeça do tronco imediatamente abaixo da mandíbula inferior (passo 1, Figura 2) usinga bisturi estéril (# 10 lâmina). Gire a cabeça para que a mandíbula está agora na placa de dissecação. Use uma pinça para estabilizar a cabeça e um bisturi para remover cerca de ¼ do lado dorsal do cérebro (passo 2, 1º corte, Figura 2) - isto é especialmente importante uma vez que a cartilagem endurece em torno e15. </li><li> Orientar a cabeça para que o bisturi pode furar o nariz entre os olhos. Fazer um corte através do centro da cabeça, de modo que os olhos estão agora em metades separadas (etapa 2, 2 nd corte, Figura 2). </li><li> Usando dois # 5 fórceps, tomar a metade da cabeça e retire o excesso de tecido (veja o passo 3, Figura 2). Certifique-se de orientar a cabeça para que a LG (localizado no canto inferior posterior do olho) não está perdido neste processo de remoção. Remover o excesso cerebral, cartilagem e tecido circundante nasal e abaixo do olho. Uma vez que a glândula é pouco visível neste momento, garantir o olho está devidamente orientado após o excesso de tecido remoção to evitar perder a localização da LG. </li><li> Cuidadosamente agarrar a pele do posterior, canto inferior do olho com as duas pinças. Retire a pele puxando cuidadosamente aberta com a pinça para expor a LG. Use iluminação adicional acima e abaixo do tecido para ajudar a visualizar o LG. Nota: A LG, distinguível por sua aparência como um botão em um duto dentro, um mesênquima condensado mais escuro, deve ser visível neste momento. </li><li> Cuidadosamente começam a dissecar a LG e mesênquima associado (veja o esquema e imagens) de distância do tecido circundante usando uma pinça, tomando cuidado para não pegar a LG ou a sua conduta de associado. Uma vez que a glândula está livre a partir do tecido circundante, remover cuidadosamente toda a glândula agarrando o epitélio circundante do olho na base da conduta de LG. Ter o cuidado de preservar a mesênquima em torno do epitélio, o que pode ser observado como um mesênquima condensada em que os invagina epitélio. NOTA: Algumas remoção adicional de tissue (quaisquer pequenos fragmentos de osso, músculo e tecido conjuntivo) pode ser necessário para evitar factores de sinalização / crescimento do tecido vizinho. </li><li> Para a cultura, a colheita 4-5 glândulas e mesênquima associado por placa (GL são banhados imediatamente após a dissecção); recolher um mínimo de 14 glândulas por 100 ul de tampão de lise de ARN para RT-PCR 6. </li></ol> 2. preparar as placas para Ex Cultura vivo Este procedimento é baseado no sistema utilizado para o desenvolvimento da glândula salivar 7,8. <ol><li> Adicionar 200 ul de DMEM F12 (suplementado com 100 U / ml de penicilina e 100 ug / ml de estreptomicina), meios de cultura com 50 ug / ml de ácido ascórbico e 50 ug / mL de holo-transferrina a um micropoço 50 milímetros de diâmetro prato de vidro de fundo 7. Nota: DMEM F12 foi escolhido como nós anteriormente achei que maximizar morfogênese na glândula salivar embrionário. </li><li> Flutuar um polycarb 13 milímetrosfiltro de membrana onate com 0,1 mm poros no topo da mídia. </li><li> Passo opcional: Diluir 3-D laminina 1: 1 com DMEM / F12 para fazer uma concentração final de 3 mg / ml (200 ul usar pipeta para misturar suavemente; centrifugar durante 1 min, se aparecerem bolhas). Adicionar 15 ul desta diluída laminina 3-D para filtrar. Nota: Esta diluição foi encontrado para ser óptima para a manutenção da LG no seu estado 3D sem comprometer a ramificação do epitélio. No entanto, não é essencial para a cultura de LG. </li><li> Colocar 4-5 LG para um filtro ou em laminina. Cultura GL ex vivo para 24-48 hr (Figura 3) ou fixar com PFA a 4% durante 20 min, para posterior análise por qPCR ou imunocoloração (Figura 4). Para a análise de qPCR de tecido glandular embrionário, consulte Rebustini et al., 2011. Para a análise de imunofluorescência de tecidos glandulares embrionárias, por favor consulte as seguintes citações: Hoffman et al, (2002) e Steinberg e.t al., (2005). </li></ol> 3. Rato pós-natal e Adultos lacrimais Glândulas (LG): Dissection <ol><li> Euthanize o mouse pós-natal de acordo com os padrões adequados da comissão ética animal. Para filhotes pós-natais dia 8 ou mais jovens, eutanásia, cortando a cabeça com uma tesoura esterilizada. Para o dia pós-natal 8 anos ou mais, spray de pele com etanol 70%. </li><li> Para localizar a LG, coloque o mouse laterais lado sob um microscópio de dissecação com iluminação de cima. Nota: O LG pós-natal e adulto estão cercados pela artéria carótida, o músculo masseter e da derme (ver Figura 5). </li><li> Com uma tesoura pequena de dissecação, fazer pequena incisão na epiderme lateralmente de onde a LG deve ser localizado. </li><li> Utilizando uma pinça, abra a epiderme em direção à orelha para expor a LG. </li><li> Use uma pinça para soltar suavemente LG do tecido circundante. </li><li> Uma vez que a LG é liberado do tecido circundante, retire cuidadosamente a LG pelo duto,por onde agarrar a conduta e o epitélio circundante do olho conectar (Figura 1). </li><li> Colocar GL em tampão de lise de ARN para RT-PCR, ou fixar com PFA a 4% durante 20 min para imunocoloração. </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. 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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>

Os autores não têm nada a revelar.

A versatilidade de cultura e manipular o LG ex vivo proporciona vantagens significativas para estudar o seu desenvolvimento. Isto inclui a velocidade a que o pesquisador é capaz de testar hipóteses e a multiplicidade de perturbações que podem ser realizados para avaliar a forma como epitelial, neuronais, células endoteliais e mesencyhmal interagem para formar o órgão. No entanto, há um número de advertências para a utilização deste modelo. Em primeiro lugar, em virtude de seu isolamento a glândula ...

A glândula lacrimal (LG) é responsável pela secreção de lágrima aquosa crítica para a acuidade visual e a saúde, a manutenção e a protecção das células da superfície ocular. LG resultados disfunção em um dos distúrbios oculares mais comuns e debilitantes: doença de olho seco deficiente aquosa, o qual é caracterizado por irritação ocular, sensibilidade à luz e uma visão diminuída. No ser humano o LG reside na órbita acima da extremidade lateral do olho onde 3-5 excretores lágrimas condutas de depósito sobre a superfície ocular. O rato tem três pares de glândulas oculares, a mais estudada de que é a glândula lacrimal (LG) anterior localizado e ventral ao ouvido (exorbital) com lágrimas que viajam para o olho através de um único canal excretor. Semelhante a outros órgãos glandulares, a LG desenvolve através do processo de morfogênese epitelial ramificação em que um único broto epitelial dentro de um mesênquima condensado passa por várias rodadas de broto e formação de duto para asou uma intrincada rede interligada de ácinos e ductos de secreção (Figura 1) 2. Durante o desenvolvimento do epitélio torna-se vascularizado, bem como fortemente inervados por nervos parassimpáticos do gânglio pterigopalatina e, em menor grau pelos nervos simpáticos do gânglio cervical superior 3. As interacções entre as células de cada um destes tipos de células neuronais ou seja, epiteliais, endoteliais e mesenquimais, são essenciais para a função e manutenção do tecido adulto. No entanto, os mecanismos moleculares subjacentes coordenação LG desenvolvimento e regeneração, bem como a forma como tipo de comunicação inter-celular orienta esses processos ainda não está claro. O advento das embrionárias ex vivo técnicas de cultura permitiu a identificação de vias de desenvolvimento e regeneração em vários órgãos de ramificação 4. A cultura ex vivo dá o pesquisador a capacidade de manipular o órgão (memecânica, química ou genética) sob condições definidas, bem como para caracterizar o desenvolvimento de órgãos e as interacções célula-célula em tempo real. A LG exorbital do mouse é altamente favorável a esta técnica e estudos recentes têm definido sistemas que regulam seu desenvolvimento 2,5 sinalização. No entanto, apesar da necessidade de entender sinais moleculares subjacentes LG desenvolvimento e regeneração, que atualmente permanece pouco estudado, provavelmente devido às dificuldades técnicas de isolamento do órgão. Neste artigo, descrevemos como isolar e executar ex vivo da cultura do embrionário murino LG para definir programas de desenvolvimento.

<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

A glândula lacrimal (LG) segrega lágrimas aquosas necessárias para a manutenção da estrutura e função da córnea, um tecido transparente essencial para a visão. No ser humano uma única LG reside na órbita acima da extremidade lateral de cada olho entregar lágrimas para a superfície ocular através de 3-5 condutas. O rato tem três pares de glândulas oculares, a mais estudada de que é a glândula lacrimal exorbital (LG) anterior localizado e ventral ao ouvido. Semelhante a outros órgãos glandulares, o LG desenvolve através do processo de epitelial morfogénese de ramificação em que um único broto epitelial dentro de um mesênquima condensada é submetido a vários ciclos de gema e formação adesiva para formar uma intrincada rede interligada de ácinos secretora e condutas. Este processo elaborado tem sido bem documentado em muitos outros órgãos epiteliais, tais como o pâncreas e glândulas salivares. No entanto, a LG tem sido muito menos explorado e os mecanismos que controlam a morfogênese são mal sobse levantou. Suspeitamos que essa sub-representação como um sistema modelo é uma conseqüência das dificuldades associadas com a descoberta, dissecando e cultivando a LG. Assim, aqui descrevemos técnicas de dissecação para a colheita embrionário e pós-natal LG e métodos para a ex vivo da cultura do tecido.

O LG desenvolve através do processo de ramificação epitelial morfogénese. Imagens de campo claro de GL embrionárias dissecados em e14, e15, e16, e17, e P2 ilustrar este evento. <img alt="A Figura 1" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig1highres.jpg" width="600" /> Figura 1. A LG desenvolve através do processo de ramificação morfogênese epitelial. Imagens de campo claro de GL embrionárias recentemente...

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Manipulating the Murine Lacrimal Gland

A glândula lacrimal (LG) é um órgão de ramificação que produz os componentes aquosos de lágrimas necessários para a manutenção da visão e saúde ocular. Aqui nós descrevemos murino LG dissecção e ex vivo de técnicas de cultura de decifrar as vias de sinalização envolvidas na LG desenvolvimento.

Manipulando o Murino lacrimais Gland

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 visualizações.  University of California San Francisco.  A glândula lacrimal (LG) é um órgão de ramificação que produz os componentes aquosos de lágrimas necessários para a manutenção da visão e saúde ocular. Aqui nós descrevemos murino LG dissecção e ex vivo de técnicas de cultura de decifrar as vias de sinalização envolvidas na LG desenvolvimento. 

Vídeo: 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.

Microchips de silício para Manipulação de célula-Interação

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

Biologia

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

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

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

Usando uma pinça Laser Para Manipulação neurônios isolados In Vitro

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

Murine Renal Transplantation Procedure

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

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

Murino Procedimento de Transplante 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.

Isolamento de Células-Tronco do rato das Glândulas Salivares

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.

Modificação genética e recombinação de culturas de órgãos de glândula salivar

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.

Injeção Intraductal para localizada Drug Delivery ao Rato glândula mamária

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.

O isolamento de células endoteliais de Murino Válvula

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.

Imunofluorescência Indireta em cortes congelados de Rato glândula mamária

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.

Isolamento de Murino vasculares coronários células musculares lisas

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.