Diese Arbeit wurde durch die Saban Research Institute Pre-Doc-Award (PMDM) unterstützt und von NIH RO1 HL75773 (DW).

Orgel Kultur ist eine sehr nützliche und vielseitige Technik zur Gen-und Proteinexpression zu untersuchen. Diese ex vivo Kultur Methoden können als Vor-oder in Ergänzung zu den in-vivo-Studien verwendet werden. 1. Embryonale Lung Isolation <ol><li> Timed-trächtigen Mäusen auf postcoitum Tag 11,5 oder 12,5 (E11.5-12.5) von CO 2 Narkose nach dem NIH und OLAW Richtlinien geopfert. Das Tier ist in einer Kammer platziert und 100% CO 2 wird eingeführt. Nachdem das Tier bewusstlos ist der CO 2-Strom wird erhöht. Klinische Tod des Tieres muss gewährleistet sein. </li><li> Alle folgenden Schritte müssen unter sterilen Bedingungen in einer Sterilbank abgeschlossen sein. Die Gebärmutter ist von der trächtigen Weibchen entfernt. Zum Entfernen der Gebärmutter aus dem Tierreich sind die trächtigen Weibchen auf den Rücken gelegt und versprüht mit 70% Ethanol. Ein Schnitt in der Bauchdecke, und die Haut wird durch Ziehen der Haut nach oben, während das Tier Hinterbeine entfernt. Die Bauchhöhle wird geöffnet und die Gebärmutter ist aus dem Tier entfernt. </li><li> Das Blut wird durch Platzierung der Gebärmutter in einer 50 ml konische Röhrchen mit kaltem Hanks Balanced Salt Solution (HBSS) gefüllt gespült, und das Rohr ist leicht für 2 Minuten rockten. </li><li> Die Gebärmutter wird dann in einer Petrischale unter dem stereoskopischen Präpariermikroskop gelegt, und die Embryonen aus der Gebärmutter durch Einschneiden der Gebärmutterwand mit Feder Schere freigegeben. Die Embryonen werden geerntet mit einem perforierten Löffel, und auf Eis in eine neue Petrischale mit HBSS. </li><li> Unter dem stereoskopischen Präpariermikroskop, sind embryonale Lunge eins nach dem anderen in eine Petrischale mit kaltem HBSS (Abbildung 1) zerlegt. </li><li> Die isolierten embryonalen Lunge ist auf Eis in eine Petrischale mit HBSS mit einer sterilen Transferpipette platziert. </li></ol> 2. Embryonale Lung Kultur <ol><li> 500 Mikroliter bis 1 Milliliter pro Vertiefung D-MEM/F-12 mit 50 Einheiten / ml Penicillin-Streptomycin ergänzt ist in einem Nunclon Polystyrol Gericht aufgenommen. </li><li> Ein Nuclepore Polycarbonat Track-Etch-Membran ist auf der Oberseite der Medien gelegt. Wenn die Nuclepore Polycarbonat Track-Etch-Membran auf der Oberseite des Media platziert wird, stellen Sie sicher, dass die glänzende Seite gegen die Medien und raue Seite nach oben zeigt ist. </li><li> Ein seziert embryonalen Lunge ist auf der Oberseite des Nuclepore Polycarbonat Track-Etch-Membran mit einer sterilen Transferpipette platziert. </li><li> Die embryonalen Lungen-Position wird mit Hilfe der Zange. Embryonale Lunge auf dem Filter gesetzt haben sollte eine intakte Luftröhre und ihre Luftröhre mit einer geraden Position gebracht werden, so dass alle Lunge zu den gleichen Innendruck haben. </li><li> Die Nunclon Polystyrol Gericht ist für die gewünschte Kultur Uhrzeit in eine Zelle Inkubator (Abbildung 2) übertragen. </li></ol> 3. Materialien 1. Embryonale Whole Lung Isolation <ol><li> Timed-schwanger (C57BL6 Wildtyp oder transgene) Mäuse auf postcoitum Tag 11,5 bis 13,5 (E11.5-13.5) geopfert werden. </li><li> Hanks &#39;Balanced Salt Solution (HBSS) (1x), flüssig, ohne Calciumchlorid, Magnesiumchlorid oder Magnesiumsulfat. (Invitrogen, Carlsbad, CA) mit 50 Einheiten / ml Penicillin-Streptomycin (Invitrogen, Carlsbad, CA) ergänzt. Bewahren Sie die HBSS bei Raumtemperatur und bei 4 ° C nach dem Öffnen. Aliquotieren und speichern Sie die Penicillin-Streptomycin bei -20 ° C. </li><li> Stereoscopic Präpariermikroskop (Leica, Wetzlar, Deutschland). </li><li> Dissection-Tools einschließlich Dumont Pinzetten, Fine Iris Schere (gerade), Noyes Frühjahr Schere und Moria ® Löffel (perforiert) (Fine Science Tools, Foster City, CA). </li></ol> 2. Embryonale ganze Lunge Kultur <ol><li> Dulbecco Modified Eagle Medium: Nutrient Mix F-12 (D-MEM/F-12) (1x), flüssig, noch 1:1 L-Glutamin, aber keine HEPES-Puffer. (Invitrogen, Carlsbad, CA) mit 50 Einheiten / ml Penicillin-Streptomycin (Invitrogen, Carlsbad, CA) ergänzt. Halten Sie die DMEM/F-12 Flasche weg vom Licht (z. B. durch Aluminiumfolie abgedeckt) und lagern bei 4 ° C. Aliquotieren und speichern Sie die Penicillin-Streptomycin bei -20 ° C. </li><li> Nuclepore Polycarbonat Track-Etch-Membran, 8,0-m, 13 mm (Whatman Nuclepore Track-Etch-Membran (Whatman, Florham Park, NJ). </li><li> Nunclon Polystyrol Schüssel mit Deckel, 4 Vertiefungen (Rochester, NY). </li><li> Einweg Transferpipetten, sterile (Fisher Scientific, Pittsburgh, PA). </li></ol><img src="/files/ftp_upload/2035/2035fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Präparation E12 embryonalen Lunge. (A) E12.5 ganze Embryo von der rechten Seite angezeigt. (B) mit der rechten Vorderbein aus dem Embryo entfernt worden ist. (C) Pinzetten von der linken Hand des Embryos stetig in die Schüssel gehalten. Die Pfeile zeigen Plan der Dissektion (D) Embryonale Lunge liegt hinter dem Herzen (entfernt) Und vor der Wirbelsäule. Diese Abbildung zeigt die Lunge nach Haut-und Herz Entfernung. (E) Pharynx Entfernung Trennung der intakten Lunge ermöglicht aus dem Embryo. (F) Überflüssige pharyngeale Gewebe und Speiseröhre wurden abgeschnitten. Dissected E12.5 embryonalen Lunge mit intakten Luftröhre und Kehlkopf wird angezeigt. (Cr) Cranial Lappen, (Med) Medial Lappen, (Ca) Schwanzflossenlappen, (Acc) Zubehör Lappen, (Le) linken Lappen. <img src="/files/ftp_upload/2035/2035fig2.jpg" alt="Abbildung 2" /> Abbildung 2. FGF9 induziert Erweiterung des Mesenchyms und der Dilatation des Epithels in der Lunge in vitro gezüchtet. (AC) E12.5 Lunge für 48 Stunden in Abwesenheit von FGF9 angebaut wird. Notieren Sie sich die Zunahme der Verzweigung. (DF) E12.5 Lunge für 48 Stunden in Anwesenheit von FGF9 gewachsen. Beachten Sie die Erweiterung des Epithel und Mesenchym bereits nach 24 Stunden Kultur (E). Nach 48 Stunden (F), ist die Wirkung auf das Epithel noch stärker ausgeprägt. Maßstab: AF 400 um 4. Der vollständige Text Protokoll für diesen experimentellen Ansatz ist in <a href="http://springerprotocols.com/Abstract/doi/10.1007/978-1-59745-019-5_5">Springer Protocols</a> .

<ol>
	<li>Alescio, T., Cassini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+in+vitro+of+tracheal+buds+by+pulmonary+mesenchyme+grafted+on+tracheal+epithelium.">Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium.</a> J Exp Zool. 150, 83-94 (1962).</li><li>Warburton, D., Schwarz, M., Tefft, D., Flores-Delgado, G., Anderson, K. D., Cardoso, W. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+lung+morphogenesis.">The molecular basis of lung morphogenesis.</a> Mech Dev. 92, 55-81 (2000).</li><li>Spooner, B. S., Hardman, P., Paulsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gravity+in+mammalian+organ+development:+differentiation+of+cultured+lung+and+pancreas+rudiments+during+spaceflight.">Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight.</a> J Exp Zool. 269, 212-222 (1994).</li><li>Moral, d. e. l., M, P., De Langhe, S. P., Sala, F. G., Veltmaat, J. M., Tefft, D., Wang, K., Warburton, D., Bellusci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+role+of+FGF9+on+epithelium+and+mesenchyme+in+mouse+embryonic+lung.">Differential role of FGF9 on epithelium and mesenchyme in mouse embryonic lung.</a> Dev Biol. 293, 77-89 (2006).</li><li>Moral, D. e. l., M, P., Sala, F. G., Tefft, D., Shi, W., Keshet, E., Bellusci, S., Warburton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-A+signaling+through+Flk-1+is+a+critical+facilitator+of+early+embryonic+lung+epithelial+to+endothelial+crosstalk+and+branching+morphogenesis.">VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis.</a> Dev Biol. 290, 177-188 (2006).</li><li>Jaskoll, T. F., Don-Wheeler, G., Johnson, R., Slavkin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+mouse+lung+morphogenesis+and+type+II+cytodifferentiation+in+serumless,+chemically+defined+medium+using+prolonged+in+vitro+cultures.">Embryonic mouse lung morphogenesis and type II cytodifferentiation in serumless, chemically defined medium using prolonged in vitro cultures.</a> Cell Differ. 24, 105-117 (1988).</li><li>Seth, R., Shum, L., Wu, F., Wuenschell, C., Hall, F. L., Slavkin, H. C., Warburton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+epidermal+growth+factor+expression+in+early+mouse+embryo+lung+branching+morphogenesis+in+culture:+antisense+oligodeoxynucleotide+inhibitory+strategy.">Role of epidermal growth factor expression in early mouse embryo lung branching morphogenesis in culture: antisense oligodeoxynucleotide inhibitory strategy.</a> Dev Biol. 158, 555-559 (1993).</li></ol>

mouse embryonic lung culture, a system to evaluate the molecular mechanisms of branching

Lung ursprüngliche Spezifikation sowie Verzweigung Morphogenese und die Bildung von verschiedenen Lungenerkrankungen Zelllinien erfordert eine spezifische Wechselwirkung der Lunge Entoderm mit seinen umliegenden Mesenchym und Mesothel. Lung Mesenchym hat sich gezeigt, dass die Quelle der induktive Signale für Lungen-Verzweigung Morphogenese werden. Epithelial-mesenchymale-mesothelial Wechselwirkungen sind auch entscheidend für die embryonale Lunge Morphogenese. Frühen embryonalen Lunge Organkultur ist ein sehr nützliches System epithelial-mesenchymale Interaktionen zu untersuchen. Beide epithelialen und mesenchymalen Morphogenese Erlös unter bestimmten Bedingungen, die leicht in das System manipuliert werden kann (in der Abwesenheit der Mutter beeinflusst und die Durchblutung). Noch wichtiger ist diese Technik kann leicht in einer serumless, chemisch definierten Kulturmedien erfolgen. Gewinn und Verlust der Funktion kann mit Hilfe exprimierten Proteine, rekombinante virale Vektoren und / oder Analyse von transgenen Maus-Stämme, Antisense-RNA, sowie RNA-Interferenz-Gen Knockdown werden.

Watch this Scientific Journal Video about Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching at JoVE.com

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Frühen embryonalen Lunge Organkultur ist ein sehr nützliches System epithelial-mesenchymale Interaktionen zu untersuchen. Beide epithelialen und mesenchymalen Morphogenese Erlös unter bestimmten Bedingungen, die leicht in das System manipuliert werden kann.

Embryonalen Maus-Lung Kultur, A-System, die molekularen Mechanismen des Branching Bewerten

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

mouse-embryonic-lung-culture-system-to-evaluate-molecular-mechanisms

Research

JoVE Journal

Biology

18.3K Aufrufe.  Childrens Hospital Los Angeles. Frühen embryonalen Lunge Organkultur ist ein sehr nützliches System epithelial-mesenchymale Interaktionen zu untersuchen. Beide epithelialen und mesenchymalen Morphogenese Erlös unter bestimmten Bedingungen, die leicht in das System manipuliert werden kann.

Serie: Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolierung und Kultivierung von Zellen aus dem Nephrogene Zone der embryonalen Maus Niere

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Forschung

Biologie

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount Immunhistochemische Analyse für Embryonale Limb Haut Gefäßsystem: ein Modellsystem zur Vascular Branching Morphogenese in Embryo-Studie

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi-Screening auf postembryonalen Phänotypen in Identifizieren C elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Ein System für<em&gt; Ex vivo</em&gt; Kultivierung von embryonalen Pankreas

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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 ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Maus embryonalen Entwicklung in einem Serum-freien Whole Embryo Culture-System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Vorgehensweise Um die Effizienz von Flockungsmittel für die Beseitigung von Dispersed Partikel aus Pflanzenextrakten zur Bewertung

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

High Content Screening-Analyse die toxikologischen Wirkungen von gefährlichen und potenziell gefährlichen Inhaltsstoffe (HPHC) zur Bewertung

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Mucin Agarose-Gel-Elektrophorese: Western Blotting für Hochmolekulare Glykoproteine


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...