Name: chick ex ovo culture and ex ovo cam assay: how it really works
Uploaded: 2009-11-30T14:45:00
Description: Watch this Scientific Journal Video about Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works at JoVE.com

De auteurs willen graag J. Kueper en J. Wittschier bedanken voor de uitstekende technische bijstand en prof. dr. Ruebben voor het leveren van ruim tumor materiaal.

Alle apparatuur en reagentia moeten worden aangeschaft steriel of moet worden warmte of stoom gesteriliseerd of gesteriliseerd met 70% EtOH. De auteurs stellen dat experimenten op dieren werden uitgevoerd in overeenstemming met de Europese Gemeenschappen richtlijn (86/609/EEG), volgens de richtlijnen van de NIH ten aanzien van de zorg en het gebruik van dieren voor experimentele procedures en de regels uiteengezet door de Institutional Animal set zorg en gebruik Comite (IACUC) aan de Univers...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+models+to+quantify+angiogenesis+induced+by+inflammatory+and+tumor+cells+or+purified+effector+molecules.">Chick embryo chorioallantoic membrane models to quantify angiogenesis induced by inflammatory and tumor cells or purified effector molecules.</a> Methods Enzymology. 444, 21-44 (2008).</li><li>Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+as+a+useful+tool+to+study+angiogenesis.">Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis.</a> Int. Rev. Cell Mol. Biol. 270, 181-224 (2008).</li><li>H&#246;ckel, M., Sasse, J., Wissler, J. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+horseradish+peroxidase+into+the+optic+nerve+after+vitreal+or+vascular+injections+in+the+developing+chick.">Penetration of horseradish peroxidase into the optic nerve after vitreal or vascular injections in the developing chick.</a> Invest Ophthalmol. Vis. Sci. 20, 705-716 (1981).</li><li>Halfter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Change+in+embryonic+eye+size+and+retinal+cell+proliferation+following+intravitreal+injection+of+glycosaminoglycans.">Change in embryonic eye size and retinal cell proliferation following intravitreal injection of glycosaminoglycans.</a> Invest. Ophthalmol. Vis. Sci. 49, 3289-3298 (2008).</li><li>Oppitz, M., Busch, C., Shriek, G., Metzger, M., Just, L., Drews, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-malignant+migration+of+B16+mouse+melanoma+cells+in+the+neural+crest+and+invasive+growth+in+the+eye+cup+of+the+chick+embryo.">Non-malignant migration of B16 mouse melanoma cells in the neural crest and invasive growth in the eye cup of the chick embryo.</a> Melanoma Res. 17, 17-30 (2007).</li><li>Uchibayashi, T., Egawa, M., Nakajima, K., Hisazumi, H., Tanaka, M., Endo, Y., Sasaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+tumour+cell+lines+implanted+onto+the+chorioallantoic+membrane+of+chick+embryo+to+anticancer+agents+in+combination+with+hyperthermia.">Responses of tumour cell lines implanted onto the chorioallantoic membrane of chick embryo to anticancer agents in combination with hyperthermia.</a> Urol. Res. 20, 237-239 (1992).</li><li>Demir, R., Naschberger, L., Demir, I., Melling, N., Dimmler, A., Papadopoulus, T., Sturzl, M., Klein, P., Hohenberger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+generates+a+more+invasive+phenotype+of+tumour+cells:+an+in+vivo+experimental+setup+based+on+the+chorioallantoic+membrane.">Hypoxia generates a more invasive phenotype of tumour cells: an in vivo experimental setup based on the chorioallantoic membrane.</a> Pathol. Oncol. Res. , (2008).</li><li>Kunzi-Rapp, K., Genze, F., Kufer, R., Reich, E., Hautmann, R. E., Gschwend, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chorioallantoic+membrane+assay:+vascularized+3-dimensional+cell+culture+system+for+human+prostate+cancer+cells+as+an+animal+substitute+model.">Chorioallantoic membrane assay: vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model.</a> J. Urol. 166, 1502-1507 (2001).</li><li>Hall, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafting+of+organs+and+tissues+to+the+chorioallantoic+membrane+of+the+embryonic+chick.">Grafting of organs and tissues to the chorioallantoic membrane of the embryonic chick.</a> TCA Manual. 4 (3), 881-883 (1978).</li><li>Kunzi-Rapp, K., Kaskel, P., Steiner, R., Peter, R. U., Krahn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+blood+levels+of+human+S100+in+melanoma+chick+embryo+xenografts'+circulation.">Increased blood levels of human S100 in melanoma chick embryo xenografts' circulation.</a> Pigment Cell Res. 14, 9-13 (2001).</li><li>McFall, R. C., Sery, T. W., Makadon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+new+continuous+cell+line+derived+from+a+human+retinoblastoma.">Characterization of a new continuous cell line derived from a human retinoblastoma.</a> Cancer Research. 37, 1003-1010 (1977).</li><li>Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+effect+of+matrix+metalloproteinase+down-regulation+on+vascular+intravasation+and+metastasis+of+human+fibrosarcoma+cells+selected+in+vivo+for+high+rates+of+dissemination.">Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination.</a> Cancer Research. 65, 10959-10969 (2005).</li><li>Kim, J., Kovalski, K., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=requirement+for+specific+proteases+in+cancer+cell+intravasation+as+revealed+by+a+novel+semiquantitative+PCR-based+assay.">requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay.</a> Cell. 94, 353-362 (1998).</li></ol>

Innovatieve of gewoon een andere chick cultuur protocol? Met de voormalige Shell-less cultuur protocollen 1-4 De totale overleving tarieven van de kippenembryo&#39;s waren laag, bijvoorbeeld ca. 30% 1. De verfijnde ex ovo cultuur protocol zoals beschreven in dit artikel video, daarentegen, maakt het mogelijk om te overleven prijzen meer dan 50%. Vergeleken met de klassieke in-ovo culturen ex ovo cultuur van kippenembryo&#39;s vergemakkelijkt a...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fertilized Eggs</td>
<td>Animal</td>
<td>S&ouml;ries Trockels, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mice</td>
<td>Animal</td>
<td>Charles Rivers Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator Type 3000 with rotating egg tray</td>
<td>Tools</td>
<td>Siepmann GmbH Germany</td>
<td>9503</td>
<td>Maintain at 37&deg;C with relative humidity set above 60%</td>
</tr>
<tr>
<td>Egg incubator BSS 160</td>
<td>Tools</td>
<td>Grumbach, Germany</td>
<td>8101</td>
<td>Maintain at 37-387deg;C with relative humidity set above 60%</td>
</tr>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>PBS should be cold (&gt; 4&deg;C) and sterile
</td>
</tr>
<tr>
<td>Dulbecco's modified eagle's medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 6429</td>
<td>DMEM culture medium</td>
</tr>
<tr>
<td>Leibovitz's L-15 Medium</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>31415-029</td>
<td>here: collection medium for tumor samples</td>
</tr>
<tr>
<td>Tumor cell -specific medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>each research group should use the medium they culture their tumors cells in or common culture media, e.g. Leibovitz's Medium or Dulbecco's modified eagle's medium supplemented with 15% fetal bovine serum (FBS), 4mM L-glutamine and 50 &mu;M insulin</td>
</tr>
<tr>
<td>70% EtOH</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic stir bar, triangled 80 mm</td>
<td>Tool</td>
<td>VWR</td>
<td>442-0391</td>
<td>A triangled magnetic stir bar serves well to crack the eggs shell.</td>
</tr>
<tr>
<td>Forceps DUMONT #5 </td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>bevelled very fine shanks (0.05 mm x 0.02 mm tip)</td>
</tr>
<tr>
<td>Forceps DUMONT #7</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11271-30</td>
<td>curved shanks (0.07 mm x 0.10 mm tip)</td>
</tr>
<tr>
<td>Spring scissors, straight, 8cm</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>fine, small straight blades</td>
</tr>
<tr>
<td>Fine Iris scissors, straight</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14094-11</td>
<td>Use to cut our the CAM</td>
</tr>
<tr>
<td>Standard scissors, straight, sharp/blunt</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14007-14</td>
<td>Use for decapitation or cervical dislocation</td>
</tr>
<tr>
<td>microliter syringe 1702 TLLX, 25 &mu;l</td>
<td>Tool</td>
<td>Hamilton</td>
<td>80222</td>
<td>Use for injection into the vitreous</td>
</tr>
<tr>
<td>Hamilton 33-G needle (15 mm)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>18073-15</td>
<td>Use for injection into the vitreous</td>
</tr>
<tr>
<td>Sterile scalpels</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use for dissecting tumor samples</td>
</tr>
<tr>
<td>Small drain spoon</td>
<td>Tool</td>
<td>Geuder, Germany</td>
<td>15758</td>
<td>Use to transfer of small chick embryos</td>
</tr>
<tr>
<td>Wax board with fixing pins</td>
<td>Tool</td>
<td>Self made</td>
<td>&nbsp;</td>
<td>Use to fix of animals for dissection</td>
</tr>
<tr>
<td>Petri dishes 60 x 15 mm</td>
<td>Tool</td>
<td>Greiner</td>
<td>628102</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes 92 x 16 mm</td>
<td>Tool</td>
<td>Sarstedt</td>
<td>82.1472</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Petri dish 100 x 20 mm</td>
<td>Tool</td>
<td>Greiner</td>
<td>664160</td>
<td>extra deep, with spacers for ventilation</td>
</tr>
<tr>
<td>Beaker</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>300-600 ml</td>
</tr>
<tr>
<td>FALCON tubes</td>
<td>Tool</td>
<td>FALCON</td>
<td>&nbsp;</td>
<td>15 ml and 50 ml</td>
</tr>
<tr>
<td>Eppendorf tubes</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>1.5 ml and 2 ml</td>
</tr>
<tr>
<td>1ml pipette tips</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use to cut plastic rings for application of substances / cells</td>
</tr>
<tr>
<td>Peripheral venous catheter (PVL)</td>
<td>Tool</td>
<td>Viggo&reg;</td>
<td>&nbsp;</td>
<td>Use to cut silicon rings from the tube for application of substances / cells</td>
</tr>
<tr>
<td>Pipettes and tips</td>
<td>Tool</td>
<td>Eppendorf / Abimed</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Autoclaved folded Filters 595 1/2, 110 mm diameter</td>
<td>Tool</td>
<td>Schleicher &amp; Schuell</td>
<td>311643</td>
<td>Carrier, which caused least irritation of the CAM; used in the paper</td>
</tr>
<tr>
<td>PTFE-Pledget, non resorbing, 6,3 x 152.4 x 1.6 mm</td>
<td>Tool</td>
<td>santec-medical</td>
<td>REF 277, LOT 832/511-1</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Hxdroxyethylcellulose Tylose H 100000</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Carrier mixed with Kollidon 17 PF; caused false positive results</td>
</tr>
<tr>
<td>Kollidon 17 PF</td>
<td>Reagent</td>
<td>BASF</td>
<td>S30200</td>
<td>mixed with Hydroxyethylcellulose; caused false positive results</td>
</tr>
<tr>
<td>Collagen Biomatrix TissuDura</td>
<td>Tool</td>
<td>Baxter Healthcare S.A.</td>
<td>REF B2246000999999</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Round glass cover slips, 11 mm</td>
<td>Tool</td>
<td>Assistent Germany</td>
<td>1001/0011</td>
<td>Carrier; causes false positive results</td>
</tr>
<tr>
<td>Bovine Collagen Sponge</td>
<td>Tool</td>
<td>Wyeth</td>
<td>&nbsp;</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Resodont Absorbable Collagen Membrane</td>
<td>Tool</td>
<td>Resorba Woundcare Germany</td>
<td>LOT 280303</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Non-Woven Swabs</td>
<td>Tool</td>
<td>Fink &amp; Walter GmbH</td>
<td>REF 328132</td>
<td>Carrier; caused false positive results</td>
</tr>		</tbody>
		</table>
		</div>

chick ex ovo culture and ex ovo cam assay: how it really works

Kip eieren in de vroege fase van de kweek zijn tussen de in vitro en in vivo systemen en zorgen voor een vasculaire testomgeving niet alleen om angiogenese studeren, maar ook voor het ontstaan ​​van tumoren te bestuderen. Na de chick chorion-membraan (CAM) heeft ontwikkeld, kan het bloedvat netwerk gemakkelijk toegankelijk is, gemanipuleerd en waargenomen en daardoor zorgt voor een optimale omgeving voor angiogenese assays. Aangezien het lymfatische systeem is niet volledig ontwikkeld tot in de late stadia van de incubatie, het kuiken embryo fungeert als een natuurlijk immunodeficiënte gastheer staat is tot duurzame geënt weefsels en cellen zonder de species-specifieke beperkingen. Naast het voeden van het ontwikkelen van allo-en xenotransplantaten, de CAM bloedvat-netwerk biedt een unieke gunstig klimaat voor tumorcel intravasation, verspreiding, en vasculaire arresteren en een repository waar gearresteerd cellen extravasaat aan micro metastatische foci te vormen. Voor experimentele doeleinden, in de meeste recente studies van de CAM werd blootgelegd door het snijden van een raam door de eischaal en de experimenten werden uitgevoerd in ovo, wat resulteert in aanzienlijke beperkingen in de toegankelijkheid van de CAM en de mogelijkheden voor observatie en documentatie van foto-effecten. Als shell-less culturen van het kippenembryo werd 1-4 gebruikt, geen experimentele details werden verstrekt en, indien gepubliceerd op alle, de overlevingskansen van deze culturen waren laag. We verfijnde de methode van ex-ovo cultuur van kippenembryo&#39;s aanzienlijk door de invoering van een rationeel gecontroleerde extrusie van de ei-inhoud. Deze ex ovo culturen verbeteren van de toegankelijkheid van de CAM en kippenembryo, zodat ze makkelijk in vivo documentatie van effecten en het vergemakkelijken van experimentele manipulatie van het embryo. Hierdoor kan de succesvolle toepassing van een groot aantal wetenschappelijke vragen: (1) Als een verbeterde angiogenese test 5,6, (2) een experimentele set-up voor vereenvoudigde injecties in het glasvocht van het kippenembryo oog 7-9, (3) als een testomgeving voor de verspreiding en intravasation van tumorcellen van gevestigde cellijnen geënt op de CAM 10-12, (4) verspreid als een verbeterd systeem voor het ondersteunen van een succesvolle transplantatie en de cultuur van de ledematen van kippen en muizen 13 als (5 ) voor transplantatie, vermeerdering, en re-enten van vaste primaire tumor weefsel verkregen van biopsies op het oppervlak van de CAM 14. In deze video artikel beschrijven we de oprichting van een verfijnde chick ex ovo cultuur-en CAM-test met overleving meer dan 50%. Daarnaast bieden wij een stap voor stap demonstratie van de succesvolle toepassing van de ex ovo cultuur voor een groot aantal wetenschappelijke toepassingen. Daniel S. Dohle, Susanne D. Pasa, en Sebastian Gustmann droegen ook voor deze studie.

Watch this Scientific Journal Video about Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works at JoVE.com

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Het kuiken chorion-membraan (CAM) is een uniek, natuurlijk immunodeficiënte ondersteunende cultuur omgeving om angiogenese en tumorigenese studie. Deze video artikel demonstreert de verschillende stappen in het kuiken<em&gt; Ex ovo</em&gt; Cultuur, de toepassing van potentieel angiogene stoffen en succesvolle inoculatie van tumor cellen en weefsels op het oppervlak van de CAM.

Kuiken Ex ovo Cultuur en Ex ovo CAM test: Hoe het echt werkt

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Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

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Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

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A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel<em&gt; Ex vivo</em&gt; Cultuur Methode voor de embryonale Mouse Hart

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

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Ex vivo cultuur van muis embryonale Huid en live-beeldvorming van melanoblast Migratie

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

An Ex vivo Culture System to Study Thyroid Development

Een<em&gt; Ex vivo</em&gt; Cultuur Systeem om Schildklier Development Study

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...