עבודה זו נתמכה על ידי פרס המחקר סבן קדם דוקטורט במכון (PMDM), ועל ידי NIH RO1 HL75773 (DW).

תרבות איברים היא טכניקה שימושית מאוד תכליתי ללמוד גנים ביטוי חלבון. תרבות שיטות אלה לשעבר vivo יכול לשמש ראשוני או משלים במחקרים vivo. 1. ריאות עובריים בידוד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מתוזמן בהריון עכברים מוקרבים ביום postcoitum 11.5 או 12.5 (E11.5 12.5-) על ידי ה-CO 2 הרדמה על פי הנחיות NIH ו OLAW. חיה ממוקם בתא ו CO 2 100% הוא הציג. לאחר שהחיה היא מודעת זרימת CO 2 הוא גדל. מוות קליני של החיה יש להבטיח. </li><li style=";text-align:right;direction:rtl"> כל השלבים הבאים צריך להסתיים בתנאים סטריליים במנדף זרימה למינרית. הרחם יוסר הנקבות בהריון. כדי להסיר את הרחם מן החיה, הנקבות בהריון ממוקמות על גבם רוססו עם אתנול 70%. מבצעים חתך באזור הבטן, העור הוסר על ידי משיכת העור כלפי מעלה תוך כדי החזקת הרגליים האחוריות של החיה. חלל הצפק נפתח הרחם יוסר החיה. </li><li style=";text-align:right;direction:rtl"> דם היא שטפה ידי הנחת הרחם צינור חרוטי 50 מ&quot;ל מלאים פתרון הנקס מלח קר מאוזן (HBSS), ואת הצינור הוא התנדנד בעדינות במשך 2 דקות. </li><li style=";text-align:right;direction:rtl"> הרחם ממוקם מכן תחת מיקרוסקופ לנתח סטריאוסקופית בצלחת פטרי, ואת העוברים שוחרר מן הרחם על ידי חריטה לדופן הרחם באמצעות מספריים האביב. עוברים נקצרים בעזרת כף מחוררת, והניחו על קרח בצלחת פטרי חדשה המכילה HBSS. </li><li style=";text-align:right;direction:rtl"> תחת המיקרוסקופ לנתח סטריאוסקופית, הריאות עובריים הם גזור אחד אחד בצלחת פטרי המכילות HBSS קר (איור 1). </li><li style=";text-align:right;direction:rtl"> ריאה עובריים מבודדים מושם על קרח בצלחת פטרי המכילות HBSS בעזרת פיפטה העברת סטרילית. </li></ol> 2. ריאות עובריים תרבות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 500 מיקרוליטר ל למיליליטר 1 לכל היטב D-MEM/F-12 בתוספת 50 יחידות / מ&quot;ל ​​של סטרפטומיצין, פניצילין הוא הוסיף בצלחת פוליסטירן Nunclon. </li><li style=";text-align:right;direction:rtl"> פוליקרבונט Nuclepore Track-Etch ממברנה ממוקם על גבי התקשורת. כאשר פוליקרבונט Nuclepore Track-Etch ממברנה ממוקם על גבי התקשורת, לוודא כי הצד המבריק הוא כלפי התקשורת, הצד המחוספס פונה מעלה. </li><li style=";text-align:right;direction:rtl"> ריאות העובר גזור ממוקמת על גבי ממברנה פוליקרבונט מסלול Etch Nuclepore באמצעות פיפטה העברת סטרילית. </li><li style=";text-align:right;direction:rtl"> עמדת ריאות העוברית מותאם באמצעות מלקחיים. ריאה עובריים שהונחו על המסנן צריך קנה הנשימה שלם להציב קנה הנשימה שלהם עם בעל עמדה ישר, ובכך מאפשר לריאות כל יש את הלחץ הפנימי זהה. </li><li style=";text-align:right;direction:rtl"> המנה פוליסטירן Nunclon מועבר בפעם תרבות הרצוי חממה תא (איור 2). </li></ol> 3. חומרים 1. עובריים ריאות שלמות בידוד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מתוזמן בהריון (C57BL6 סוג בר או מהונדס) עכברים להיות הקריב ביום postcoitum 11.5-13.5 (E11.5-13.5). </li><li style=";text-align:right;direction:rtl"> הנקס &quot;פתרון מאוזן מלח (HBSS) (1X), נוזלי, ללא כלור, סידן, מגנזיום כלוריד, או מגנזיום סולפט. (Invitrogen, בקרלסבד, קליפורניה) בתוספת 50 יחידות / מ&quot;ל ​​של סטרפטומיצין, פניצילין (Invitrogen, בקרלסבד, קליפורניה). חנות HBSS בטמפרטורת החדר ב 4 ° C לאחר הפתיחה. Aliquot ולאחסן את הפניצילין, סטרפטומיצין ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> מיקרוסקופ לנתח סטריאוסקופית (לייקה, Wetzlar, גרמניה). </li><li style=";text-align:right;direction:rtl"> Dissection כלים כולל דומון מלקחיים, מספריים איריס פיין (ישר), נויס באביב מספריים מוריה ® כפית (מחורר) (כלי מדעי בסדר, פוסטר סיטי, קליפורניה). </li></ol> 2. עובריים ריאות שלמות תרבות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתנה Dulbecco הנשר בינונית: מערבבים מזין F-12 (D-MEM/F-12) (1X), נוזל, 01:01 מכיל L-גלוטמין, אבל שום חיץ HEPES. (Invitrogen, בקרלסבד, קליפורניה) בתוספת 50 יחידות / מ&quot;ל ​​של סטרפטומיצין, פניצילין (Invitrogen, בקרלסבד, קליפורניה). שמור את הבקבוק DMEM/F-12 מן האור (למשל על ידי מכוסה בנייר אלומיניום) ולאחסן ב 4 ° C. Aliquot ולאחסן את הפניצילין, סטרפטומיצין ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> Nuclepore פוליקרבונט Track-Etch ממברנה, 8.0-מ &#39;, 13 מ&quot;מ (Whatman Nuclepore Track-Etch קרום (Whatman, Florham Park, NJ). </li><li style=";text-align:right;direction:rtl"> Nunclon צלחת פוליסטירן עם מכסים, 4 בארות (רוצ&#39;סטר, ניו יורק). </li><li style=";text-align:right;direction:rtl"> Pipettes העברת פנויה, סטרילי (פישר סיינטיפיק, פיטסבורג, פנסילבניה). </li></ol><img src="/files/ftp_upload/2035/2035fig1.jpg" alt="איור 1" /> באיור 1. Dissection של E12 הריאות עובריים. (א) E12.5 העובר כולו במבט מהצד הנכון. (ב) forelimb ימין הוסר העובר. (C) מלקחיים המוחזקות על ידי יד שמאל מחזיקה את העובר קבוע בצלחת. החצים מצביעים על תוכנית של דיסקציה (ד &#39;) ריאות עובריים טמונה האחורי ללב (מוסר) ו הקדמי לעמוד השדרה. נתון זה מראה את הריאות לאחר הסרת העור לב. (ה) הסרת הלוע מאפשרת הפרדה של הריאה שלמים מן העובר. (ו) רקמת הלוע והוושט חיצוניים ואת כבר גזוז משם. גזור E12.5 ריאות העובר עם קנה הנשימה הגרון שלם מוצג. (CR) באונה גולגולתי, (מד) באונה המדיאלי, (CA) באונה הזנב, (ACC) באונה אביזרים, (לה) באונה השמאלית. <img src="/files/ftp_upload/2035/2035fig2.jpg" alt="איור 2" /> איור 2. FGF9 גורם להרחבת mesenchyme ועל התרחבות של האפיתל בריאה לגדל במבחנה. (AC) E12.5 ריאות הוא גדל במשך 48 שעות העדר FGF9. הערה העלייה הסתעפות. (DF) E12.5 הריאות גדל במשך 48 שעות הנוכחות של FGF9. הערה התרחבות של האפיתל וגם mesenchyme מוקדם ככל לאחר 24 שעות של תרבות (E). לאחר 48 שעות (F), ההשפעה על האפיתל הוא מודגש עוד יותר. סולם בר: AF 400 מיקרומטר 4. פרוטוקול טקסט מלא לגישה זו ניסיוני זמין <a href="http://springerprotocols.com/Abstract/doi/10.1007/978-1-59745-019-5_5">הפרוטוקולים שפרינגר</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>

אין ניגודי אינטרסים הכריז.

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

מפרט ריאות בראשיתי כמו גם המורפוגנזה הסתעפות ויצירת שושלות תאים שונים ריאתי דורש אינטראקציה ספציפית של האנדודרם הריאה עם mesenchyme המקיפים שלה mesothelium. ריאות mesenchyme הוכח להיות מקור של אותות אינדוקטיבי עבור המורפוגנזה ריאות הסתעפות. אפיתל-mesenchymal-mesothelial אינטראקציות גם קריטי המורפוגנזה ריאות העובר. איברים עובריים מוקדמים תרבות ריאות היא מערכת שימושית מאוד ללמוד אפיתל-mesenchymal אינטראקציות. המורפוגנזה ההכנסות הן אפיתל mesenchymal בתנאים מסוימים, כי ניתן להשפיע בקלות במערכת זו (בהיעדר השפעה אימהית זרימת הדם). וחשוב יותר טכניקה זו ניתן לעשות זאת בקלות, התקשורת serumless תרבות הגדרה כימית. רווח והפסד של פונקציה יכולה להיות מושגת באמצעות חלבונים לידי ביטוי, וקטורים ויראליים רקומביננטי ו / או ניתוח של זנים עכבר מהונדס, antisense RNA, כמו גם גנים RNA מציאה הפרעה.

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

איברים עובריים מוקדמים תרבות ריאות היא מערכת שימושית מאוד ללמוד אפיתל-mesenchymal אינטראקציות. המורפוגנזה ההכנסות הן אפיתל mesenchymal בתנאים מסוימים, כי ניתן להשפיע בקלות במערכת זו.

עכבר עובריים תרבות ריאות, מערכת הערכת המנגנונים המולקולריים של מסעף

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

Nanyang Technological University

Research

JoVE Journal

Biology

18.4K Views.  Childrens Hospital Los Angeles. איברים עובריים מוקדמים תרבות ריאות היא מערכת שימושית מאוד ללמוד אפיתל-mesenchymal אינטראקציות. המורפוגנזה ההכנסות הן אפיתל mesenchymal בתנאים מסוימים, כי ניתן להשפיע בקלות במערכת זו.

Video: 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.

בידוד והתרבות של תאים מאזור nephrogenic של הכליה עכבר עובריים

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

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 עבור ניתוח immunohistochemical vasculature לימב עובריים עור: מערכת מודל לחקר המורפוגנזה מסעף דם בעובר

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

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

ויזואליזציה של Caenorhabditis elegans לציפורן מבנים באמצעות דיי ויטל lipophilic, DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

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 הקרנת לזהות פנוטיפים Postembryonic ב ג 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.

מערכת ל<em&gt; Vivo לשעבר</em&gt; Culturing של לבלב עוברי

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.

Explants Blastomere לבדיקת מחויבות גורל תא במהלך התפתחות עוברית

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.

רומן<em&gt; Ex vivo</em&gt; שיטת תרבות ללב העכבר העוברית

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.

עכבר עוברי פיתוח במערכת סרום ללא תרבות שלמה עובר

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

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

בידוד והתרבות ראשית של לתאי אנדותל עכבר אב העורקים

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

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.

נוהל כדי להעריך את היעילות של flocculants להרחקת מפוזרי חלקיקים מן תמציות צמחים

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 ...

עכבר עובריים תרבות ריאות, מערכת הערכת המנגנונים המולקולריים של מסעף

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