Agradecemos Erika Mileti para excelente suporte técnico e Dr. Antonio Di Sabatino para fornecer as câmaras de oxigênio. Financiamento: Este trabalho foi financiado por doações do sétimo programa-quadro da UE (IBDase, ERC: Dendroworld) e Fundação Cariplo para MR e suporte por um Curie rede de mobilidade internacional de formação Marie (Cross-Talk, subvenção No: 21553-2) para KT.

1. Obtenção e Preparação do tecido <ol><li> Tissue (saudável ou IBD mucosa) é obtido durante a cirurgia. Quando a amostra é excisada transferência para o laboratório, logo que possível, mantendo-o em Ca + + / Mg + + tampão HBSS suplementado com Pen / Strep a 4 ° C ou sobre gelo. </li></ol> * O tamanho da amostra depende da disponibilidade do tecido: a parte do tecido obtido, é que não é estritamente necessária para o diagnóstico e pode variar bastante entre indivíduos saudáveis ​​e IBD. Tecido sadio é retirado de pacientes submetidos à cirurgia de câncer de cólon. <ol start="2"><li> Lave o tecido com cuidado e limpe-o de todo o material mesentérica. Separa-se a camada mucosa a partir da submucosa utilizando tesouras e pinças esterilizadas, mantendo o tecido em tampão HBSS numa placa de Petri (não necessariamente imersas). Tocar a superfície da mucosa com a pinça tão pouco quanto possível. </li><li> Corte mucosa em listras pelo menos um cm de largura e tanto tempo quanto possível. Utilize dois bisturis estéreis, tanto quanto se estivesse cortando seu alimento. </li><li> Lavar limpar suavemente mucosa em HBSS e estendê-la numa placa de Petri, com o lado apical voltada para cima. </li></ol> 2. Montagem do Tissue <ol><li> Prepare meio fresco (tisDMEM). Use DMEM suplementado com glutamina (2 mM) e de NaPyr (1 mM), e adicionar 15% de FBS-Na (Fetal Bovine Serum - norte-americana), 1% de ITS-X e de 200 ng / ml de EGF, no momento da utilização. </li><li> Encha de 10 cm de uma placa de Petri com o resto da SBF-Na (cerca de 30 ml de utilizar de modo a que as redes de metal será completamente submersa) e submergir as grelhas de metal. Mantenha a placa de abrir e apoiar os cilindros de plástico em sua torneira. </li></ol> * As grades de metal são feitos de ferro. A malha é de forma quadrada com um lado de 0,5 mm. <ol start="3"><li> Aqui 3 ul de cola cirúrgica com uma pipeta de 10 ou 20 ul. Aplique-o cuidadosamente a uma das fronteiras de um cilindro de plástico, mantendo-fIRM com a pinça. </li><li> Suavemente colocar o cilindro no lado apical da mucosa, tal como próximo de uma das bordas da tira possível. </li><li> Para se obter o tempo de cola para fixar firmemente o cilindro sobre o tecido, se preparar a placa de cultura de órgãos de centro bem: Pipetar 850 mL-1 ul do meio no centro do poço e apoiar a grade de metal nas bordas da cavidade central da placa. </li><li> Corte o excesso de tecido das bordas do cilindro usando dois bisturis estéreis como antes. </li><li> Suavemente levantar o cilindro com o tecido ligado e colocar o lado basolateral da grelha de metal no centro do prato. </li></ol> 3. Estimulação <ol><li> Use os estímulos de sua escolha em sua concentração preferido. </li><li> Aplicar volume apropriado no centro do cilindro de plástico, sem perturbar o cilindro ou a superfície da mucosa com a ponta da pipeta. Verifique se o seu volume de escolhido cobre uniformemente a superfície interna do cilindro. Emvolumes dicated: Cilindro de plástico grande: 200 ul Médio plástico do cilindro: 100 mL Cilindro de plástico de pequeno porte: 50 ul </li><li> Incubar por até 3 horas em uma incubadora convencional. </li></ol> * Se você deseja incubar por momentos mais longos, certifique-se de usar um volume muito menor de estímulos (10-20 mL) e coloque diretamente no tecido em uma atmosfera de 100% O 2 na pressão de 1 atm (ver 3.5) . <ol start="4"><li> Tome o meio com os estímulos fora da superfície apical do tecido e não substitui com meio fresco, porque o tecido não se cansa O 2 se uma espessa camada de meio na face apical impede a troca gasosa. Cubra o prato. </li><li> Colocar o tecido numa atmosfera de 100% de O2 na pressão de 1 atm. </li></ol> 4. Colheita <ol><li> Depois de 24 horas de tempo de cultivo total (± 2,5 hr) retire cuidadosamente o cilindro a partir de diade tecido e com a ajuda de um escalpelo cola raspando suavemente e armazená-lo em função da análise desejada (corrigir para imuno-histoquímica e / ou ensaios de imunofluorescência, encaixe ou por congelamento em N2 líquido para purificação de RNA e de perfis de expressão de genes ou a purificação de proteínas e ensaios de Western blot ). </li><li> Colha o meio basolateral para o perfil de secreção de citocinas. Centrifugar a 8000 rpm durante 7 min para detritos da pelota, transferir o sobrenadante num tubo Eppendorf (alternativamente aliquota) imediatamente e armazenar a -20 ° C. </li></ol> * Se você quiser manter sobrenadantes por mais tempo, armazená-los à temperatura de -80 ° C.

<ol>
	<li>Schuller, S., Lucas, M., Kaper, J. B., Giron, J. A., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ex+vivo+response+of+human+intestinal+mucosa+to+enteropathogenic+Escherichia+coli+infection.">The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection.</a> Cell. Microbiol. 11, 521-530 (2009).</li><li>Tsilingiri, K., 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=Probiotic+and+postbiotic+activity+in+health+and+disease:+comparison+on+a+novel+polarised+ex-vivo+organ+culture+model.">Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.</a> Gut. , (2012).</li><li>Tsilingiri, K., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+probiotics+be+tested+on+ex+vivo+organ+culture+models?.">Should probiotics be tested on ex vivo organ culture models?.</a> Gut Microbes. , (2012).</li><li>Mileti, E., Matteoli, G., Iliev, I. D., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+immunomodulatory+properties+of+three+probiotic+strains+of+Lactobacilli+using+complex+culture+systems:+prediction+for+in+vivo+efficacy.">Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy.</a> PLoS One. 4, e7056 (2009).</li><li>Cencic, A., Langerholc, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cell+models+of+the+gut+and+their+applications+in+food+microbiology--a+review.">Functional cell models of the gut and their applications in food microbiology--a review.</a> Int. J. Food Microbiol. 141, S4-S14 (2010).</li><li>te Velde, A. A., Verstege, M. A., Hommes, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+current+practice+in+murine+TNBS-induced+colitis.">Critical appraisal of the current practice in murine TNBS-induced colitis.</a> Inflamm. Bowel Dis. 12, 995-999 (2006).</li><li>Senior, P. V., Pritchett, C. J., Sunter, J. P., Appleton, D. R., Watson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crypt+regeneration+in+adult+human+colonic+mucosa+during+prolonged+organ+culture.">Crypt regeneration in adult human colonic mucosa during prolonged organ culture.</a> J. Anat. 134, 459-469 (1982).</li><li>Browning, T. H., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+culture+of+mucosal+biopsies+of+human+small+intestine.">Organ culture of mucosal biopsies of human small intestine.</a> J. Clin. Invest. 48, 1423-1432 (1969).</li><li>Besselink, M. G., 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=Probiotic+prophylaxis+in+predicted+severe+acute+pancreatitis:+a+randomised,+double-blind,+placebo-controlled+trial.">Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial.</a> Lancet. 371, 651-659 (2008).</li><li>Lakhdari, O., 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=Identification+of+NF-kappaB+modulation+capabilities+within+human+intestinal+commensal+bacteria.">Identification of NF-kappaB modulation capabilities within human intestinal commensal bacteria.</a> J. Biomed. Biotechnol. 2011, 282356 (2011).</li></ol>

Não há conflitos de interesse declarados.

A necessidade de modelos fisiologicamente relevantes em que os tratamentos para mucosa intestinal humana pode ser validamente testados tem sido enfatizada. Muitos pesquisadores têm identificado potenciais artefatos e defeitos tanto na célula 5 e 6 modelos de mouse usado até agora para esse fim. Na verdade, mesmo que os dados pré-clínicos promissores é muitas vezes obtido, estes raramente se traduzem em benefícios clínicos significativos. O método descrito neste...

<table border="1">
	<tbody>
		<tr>
			<td>Regeant</td>
			<td>Company</td>
			<td>Catalog No.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>10X HBSS</td>
			<td>Eurocalne</td>
			<td>ECM4006XL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peni/Strepto</td>
			<td>Lonza</td>
			<td>DE17602E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gibco</td>
			<td>15750-037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12614</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS-Na</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100X ITS-X</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Tebu-bio</td>
			<td>100-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17605E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Non essential aminoacids 100X</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaPyr</td>
			<td>Gibco</td>
			<td>11360-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Cloning cylinders 6x8 mm</td>
			<td>BellCo</td>
			<td>2090-00608</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 8x8 mm</td>
			<td>BellCo</td>
			<td>2090-0080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 10x10 mm</td>
			<td>BellCo</td>
			<td>2090-01010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Metal grids</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centre well organ culture plate</td>
			<td>BD Falcon</td>
			<td>353037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical glue (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen Jars</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen tanks</td>
			<td>Air Liquid Sanit&agrave;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a novel method for the culture and polarized stimulation of human intestinal mucosa explants

Existem poucos modelos atualmente para simular realisticamente micro-ambiente do complexo do intestino humano, onde uma variedade de interações ocorrem. Homeostase adequada depende directamente estas interacções, visto que todo modelar uma resposta imunológica induzir tolerância contra os antigénios dos alimentos, enquanto ao mesmo tempo de montagem respostas imunes eficazes contra micróbios patogénicos acidentalmente ingeridas com alimentos. Homeostase intestinal também é preservada através de várias interacções complexas entre a microbiota (incluindo as estirpes bacterianas benéficas associadas ao alimento) e do hospedeiro, que regulam a fixação / degradação do muco, da produção dos péptidos antimicrobianos, através da barreira epitelial, e a &quot;formação&quot; do células epiteliais &quot;, que controla o fenótipo tolerogenic ou imunogênica de únicas, gut-residentes células linfóides das populações. Estas interacções têm sido muito difíceis de reproduzir com ensaios in vitroutilizando quer linhas de células cultivadas ou de células mononucleares do sangue periférico. Além disso, os modelos de rato diferem substancialmente em componentes da mucosa intestinal (organização da camada de muco, a comunidade de bactérias comensais) com respeito ao intestino humano. Assim, os estudos de uma grande variedade de tratamentos para ser interpostos nas clínicas para as condições relacionadas com o stress ou patológicos importantes, tais como a síndrome do intestino irritável, doença inflamatória do intestino ou o cancro colo-rectal têm sido difíceis de realizar. Para tratar dessas questões, foi desenvolvido um novo sistema que nos permite estimular explantes de mucosa intestinal humana, que mantêm o seu in situ condicionado pela microbiota de acolhimento e resposta imune, de forma polarizada. Estimulação apical polarizada é de grande importância para o resultado da resposta imunitária induzida. Tem sido repetidamente demonstrado que os mesmos estímulos podem produzir respostas completamente diferentes quando ignorar a cara apical do epi intestinalthelium, estimulando as células epiteliais basolateralmente ou entrar em contato direto com os componentes da lâmina própria, mudando o fenótipo de tolerogenic para imunogênica e causando inflamação excessiva e desnecessária na área. Conseguimos estimulação polarizada por colagem de um cilindro caverna que a área delimitada da estimulação na cara apical da mucosa, como será descrito no protocolo. Nós usamos este modelo para examinar, entre outros, os efeitos diferenciais de três estirpes diferentes de bactérias lácticas. Mostramos que este modelo de sistema é muito poderoso para avaliar as propriedades imunomoduladoras de probióticos em condições saudáveis ​​e doença.

Conseguimos manter a mucosa intestinal humana saudável e IBD em cultura por pelo menos 24 horas preservando o bem-estar do tecido. Em conformidade com as observações anteriores 1, isto foi apenas possível se a maior parte do tempo de cultura (de pelo menos 85% do total) foi realizada em O 2, tal como o tecido não sobreviveram durante 24 horas em incubadoras convencionais (Figura 2). Mostramos também que as diferentes respostas de explantes pode ser observado neste modelo, dep...

Watch this Scientific Journal Video about A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants at JoVE.com

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Introduzimos um novo método para a manutenção da mucosa intestinal humana em cultura e monitorização da resposta a vários tipos de estímulos durante pelo menos 24 horas. Com o nosso método, a polaridade do tecido é mantida, o que permite um estímulo fisiológico por via apical.

Um novo método para a Cultura e polarizada Estimulação dos Humanos explantes mucosa intestinal

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

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Research

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Biology

13.0K visualizações.  European Institute of Oncology. Introduzimos um novo método para a manutenção da mucosa intestinal humana em cultura e monitorização da resposta a vários tipos de estímulos durante pelo menos 24 horas. Com o nosso método, a polaridade do tecido é mantida, o que permite um estímulo fisiológico por via apical.

Vídeo: A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para a Cultura de Embriões início pintainho ex vivo (Nova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biologia

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

Preparação e Cultura de explantes Rat Lens epitelial para estudar a diferenciação terminal

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

O método de cultura de embriões de roedores inteiro usando o Rotator do tipo Sistema de Cultura Garrafa

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Explantes blastômero para testar Compromisso destino celular durante o desenvolvimento embrionário

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de Cultura para o Rato coração embrionário

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

O método de cultura fatia para acompanhar o desenvolvimento de germes dentários Em Explant Cultura

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Subclonagem Além disso Inserção (SPI) - Um Método recombineering Novel para a construção rápida de Gene Segmentação Vetores

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Um Protocolo de Transdução Lentivirus e Análise de Downstream da Intestinal Organóides

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Um método para selecionar Aptamers de comutação Estrutura Aplicado a um colorimétrico nanopartículas de ouro Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Método cryosectioning para Microdissection de Murino Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...