Wir danken Erika Mileti für hervorragende technische Unterstützung und Dr. Antonio Di Sabatino für die Bereitstellung der Sauerstoff Kammern. Finanzierung: und Fondazione Cariplo zu MR und Unterstützung durch einen internationalen Marie-Curie Training Mobilität Netzwerk (Cross-Talk, Finanzhilfevereinbarung Nr.: 21553-2) an: Diese Arbeit wurde durch Zuschüsse aus dem 7. EU-Rahmenprogramm (Dendroworld IBDase, ERC) unterstützt KT.

1. Beziehen und Vorbereiten des Tissue <ol><li> Tissue (gesund oder IBD Schleimhaut) wird während der Operation erhalten. Sobald die Probe Transfer zum Labor herausgeschnitten so bald wie möglich, indem sie es in Ca + + / Mg + +-freiem HBSS-Puffer mit Pen / Strep bei 4 ° C oder auf Eis ergänzt. </li></ol> * Die Größe der Probe hängt von der Verfügbarkeit des Gewebes: die Teil des Gewebes erhalten, ist streng, was nicht für die Diagnose benötigt und kann stark variieren zwischen gesund und IBD Themen. Gesundes Gewebe wird von Patienten, die operiert für Darmkrebs ausgeschnitten. <ol start="2"><li> Waschen Sie das Gewebe schonend und reinigen aller mesenterialen Material. Trennen der Schicht von der Schleimhaut Submukosa mit sterilen Scheren und Zangen, während das Gewebe in HBSS-Puffer in einer Petrischale (nicht getaucht). Berühren Sie die Oberfläche der Schleimhaut mit der Zange so wenig wie möglich. </li><li> Cut Schleimhaut in Streifen mindestens 1 cm breit und so lang wie möglich. Verwenden Sie zwei sterile Skalpelle, viel, als ob Sie Ihre Lebensmittel schneiden wurden. </li><li> Waschen Sie reinigen sanft Schleimhaut in HBSS und erweitern sie auf einer Petrischale mit der apikalen Seite nach oben zeigt. </li></ol> 2. Montage des Tissue <ol><li> Bereiten Sie frisches Medium (tisDMEM). Verwenden DMEM mit Glutamin (2 mM) und NaPyr (1 mM) ergänzt, und fügen Sie 15% FBS-Na (Fetal Bovine Serum - North American), 1% ITS-X und 200 ng / ml EGF zum Zeitpunkt der Verwendung. </li><li> Füllen Sie eine 10 cm Petrischale mit dem Rest der FBS-Na (verwenden Sie etwa 30 ml, so dass Metallgitter vollständig untergetaucht werden) und tauchen die Metallgitter. Halten Sie die Platte öffnen und unterstützt die Kunststoff-Zylinder auf seinem Hahn. </li></ol> * Die Metallgitter sind aus Eisen hergestellt. Das Netz ist quadratisch mit einer Seitenlänge von 0,5 mm. <ol start="3"><li> Take 3 ul chirurgische Kleber mit einem 10 oder 20 ul Pipette. Wenden Sie es sorgfältig, um eine der Grenzen eines Kunststoff-Zylinder, während es fIRM mit der Pinzette. </li><li> Sanft legen den Zylinder auf der apikalen Seite der Schleimhaut möglichst nahe an einem der Ränder des Streifens wie möglich. </li><li> Um die Zeit zu geben, kleben fest an den Zylinder auf dem Gewebe, bereiten Sie das Zentrum gut Organkultur Platte: Dosieren 850 ul-1 ml Medium im Zentrum gut und unterstützen die Metallgitter an den Kanten der Platte zentralen gut. </li><li> Schneiden Sie überschüssiges Gewebe von den Grenzen des Zylinders mit zwei sterile Skalpelle wie zuvor. </li><li> Heben Sie den Zylinder mit dem beigefügten Gewebe und platzieren Sie den basolateralen Seite auf dem Metallgitter in der Mitte der Platte. </li></ol> 3. Stimulation <ol><li> Verwenden Sie die Reize Ihrer Wahl in Ihre bevorzugte Konzentration. </li><li> Tragen Sie geeignete Volumen in der Mitte des Kunststoff-Zylinder ohne die Zylinder oder die Schleimhaut mit der Pipettenspitze. Stellen Sie sicher, dass Ihre gewählte Lautstärke gleichmäßig bedeckt die Oberfläche im Inneren des Zylinders. Indicated Bände: Große Kunststoff-Zylinder: 200 ul Medium Kunststoff-Zylinder: 100 ul Kleine Kunststoff-Zylinder: 50 ul </li><li> Inkubieren für 3 Stunden in einem üblichen Brutschrank. </li></ol> * Wenn Sie sich für längere Zeit Punkte brüten wollen, verwenden Sie unbedingt einen viel kleineren Volumen von Reizen (10-20 ul) und direkt zu platzieren das Gewebe in einer Atmosphäre von 100% O 2 in der Druck von 1 Atm (siehe 3.5) . <ol start="4"><li> Nehmen Sie das Medium mit den Reizen aus der apikalen Oberfläche des Gewebes und nicht mit frischem Medium REPLACE, weil das Gewebe nicht genug bekommen O 2, wenn eine dicke Schicht des Mediums auf der apikalen Gesicht verhindert Gasaustausch. Cover der Platte. </li><li> Legen Sie das Gewebe in einer Atmosphäre von 100% O 2 in der Druck von 1 Atm. </li></ol> 4. Ernten <ol><li> Nach 24 Stunden der gesamten Kulturzeit (± 2,5 Stunden) vorsichtig den Zylinder von the Gewebe mit Hilfe eines Skalpells leichtem Schaben Klebstoff aus und speichert sie in Abhängigkeit von der gewünschten Analyse (fix für die Immunhistochemie und / oder Immunfluoreszenz oder Schnellfrostmedium in flüssigem N2 für die RNA-Reinigung und Genexpressionsanalyse oder Proteinreinigung und Western-Blot-Assays ). </li><li> Ernten Sie die basolateralen Medium für Cytokinsekretion Profilierung. Zentrifuge bei 8.000 rpm für 7 min zur Pelletierung Schutt, den Überstand in ein Eppendorf-Röhrchen (alternativ Aliquot) und sofort bei -20 ° C lagern </li></ol> * Wenn Sie Überstände länger behalten möchten, speichern Sie sie bei -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>

Keine Interessenskonflikte erklärt.

Der Bedarf an physiologisch relevanten Modelle, auf denen Behandlungen für den menschlichen Darmschleimhaut wirksam getestet werden seit langem betont. Viele Forscher haben potentielle Artefakte und Defekte in den beiden Zellen 5 und 6 Mausmodellen bisher für diesen Zweck verwendet identifiziert. In der Tat, obwohl vielversprechende präklinische Daten oft erhalten wird, diese nur selten zu signifikanten klinischen Nutzen übersetzen. Das Verfahren in dieser Arbeit be...

<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

Wenige Modelle existieren derzeit realistisch zu simulieren, die komplexe menschliche Darm Mikro-Umgebung, in der eine Vielzahl von Interaktionen stattfinden. Proper Homöostase hängt direkt von dieser Wechselwirkungen, wie sie eine ganze Immunantwort induzieren Toleranz gegen Nahrungsmittel-Antigene zu gestalten, während gleichzeitig Montage effektive Immunantwort gegen pathogene Mikroben versehentlich mit der Nahrung aufgenommen. Intestinale Homöostase wird auch durch verschiedene komplexe Wechselwirkungen zwischen dem microbiota (einschließlich Lebensmittel-assoziierte positive Bakterienstämme) und der Gastgeber, die die Anlage / Abbau von Schleim regulieren erhalten, die Produktion von antimikrobiellen Peptiden durch die epithelialen Barriere, und die &quot;Erziehung&quot; der Epithelzellen Bevölkerungen, die die tolerogenen oder immunogen Phänotyp der einzigartigen, gut-resident Lymphoidzellen steuert &quot;. Diese Wechselwirkungen sind bisher sehr schwierig, mit in-vitro-Assays zu reproduzieren gewesenentweder mit kultivierten Zelllinien oder peripheren mononukleären Blutzellen. Darüber hinaus unterscheiden sich im Wesentlichen in Mausmodellen Komponenten der Darmschleimhaut (Schleimschicht Organisation, symbiotischer Bakterien Gemeinde) in Bezug auf den menschlichen Darm. So haben Studien von einer Vielzahl von Anwendungen in der Klinik für wichtige stressbedingten oder pathologischer Zustände, wie Reizdarmsyndrom, entzündliche Darmerkrankung oder Darmkrebs gebracht schwierig durchzuführen. Um diese Probleme anzugehen, haben wir ein neues System, das uns zu den Explantaten der menschlichen Darmschleimhaut, die ihre in situ Konditionierung behalten durch den Host Mikroflora und Immunantwort zu stimulieren, in einer polarisierten Mode ermöglicht. Polarized apikalen Stimulation ist von großer Bedeutung für das Ergebnis der ausgelösten Immunantwort. Es hat sich wiederholt gezeigt, dass die gleichen Reize können ganz unterschiedliche Reaktionen hervorrufen, wenn sie das Gesicht der apikalen Darm epi umgehenEndothel, anregend Epithelzellen basolateral oder in direkten Kontakt mit Lamina propria Komponenten, Schalt den Phänotyp von tolerogenen um immunogen und unnötige und exzessive Entzündung in der Umgebung. Wir erzielten polarisierten Stimulation durch Kleben eine Höhle Zylinder begrenzt die Fläche der Stimulation auf der apikalen Seite der Schleimhaut im Protokoll beschrieben. Wir haben dieses Modell zu prüfen, unter anderem, unterschiedliche Auswirkungen von drei verschiedenen Laktobazillen-Stämme. Wir zeigen, dass dieses Modell sehr leistungsfähiges System, um die immunmodulatorischen Eigenschaften von Probiotika bei gesunden und Krankheitszuständen beurteilen.

Es gelang uns bei der Erhaltung gesunder und IBD menschlichen Darmschleimhaut in Kultur für mindestens 24 Stunden die Erhaltung des Wohlbefindens des Gewebes. In Übereinstimmung mit früheren Beobachtungen 1 war dies nur möglich, wenn die Mehrheit der Kultur der Zeit (mindestens 85% der Gesamtmenge) fand im O 2, wenn das Gewebe nicht für 24 Stunden in herkömmlichen Inkubatoren (Abbildung 2) zu überleben. Wir haben auch gezeigt, dass die verschiedenen Reaktionen der Explantate...

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

Wir stellen ein neues Verfahren für die Pflege der menschlichen Darmschleimhaut in Kultur und Überwachung der Reaktion auf verschiedene Arten von Stimuli über mindestens 24 Stunden. Mit unserer Methode wird die Polarität des Gewebes aufrechterhalten wird, so dass für eine physiologische Stimulation über die apikale Route.

Eine neue Methode für die Kultur und polarisierte Stimulation der menschlichen Darmschleimhaut Explantate

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

a-novel-method-for-culture-polarized-stimulation-human-intestinal

Research

JoVE Journal

Biology

13.0K Aufrufe.  European Institute of Oncology. Wir stellen ein neues Verfahren für die Pflege der menschlichen Darmschleimhaut in Kultur und Überwachung der Reaktion auf verschiedene Arten von Stimuli über mindestens 24 Stunden. Mit unserer Methode wird die Polarität des Gewebes aufrechterhalten wird, so dass für eine physiologische Stimulation über die apikale Route.

Serie: 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.

Method for Culture of Early Hühnerembryonen 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 ...

Forschung

Biologie

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.

Vorbereitung und Kultur der Rat Linsenepithelzellen Explantate für ein Studium der terminalen Differenzierung

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.

Die Methode der Nager Whole Embryo Culture mit der Rotator-type Bottle Kultur-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 ...

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.

Blastomere Explantate für das Schicksal der Zelle Engagement während der embryonalen Entwicklung Testen

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; Kultur Verfahren zur embryonalen Maus Herz

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.

Die Slice-Kultur-Verfahren zur Nach Entwicklung der Zahnkeime In Explantation Kultur

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.

Subklonierung Plus-Insertion (SPI) - A Novel Recombineering Verfahren zur schnellen Bau der Gene Targeting Vektoren

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.

Ein Protokoll für Lentivirale Transduktion und Downstream-Analyse der Darm Organoide

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.

Ein Verfahren zur Auswahl Struktur-Schalt Aptamere zu einem Farbmetrik Gold-Nanopartikeln Assay angewandt

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.

Kryoschneiden Verfahren zur Mikrodissektion von Murine Kolonschleimhaut

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