Name: the in ovo cam-assay as a xenograft model for sarcoma
Uploaded: 2013-07-17T21:00:00
Description: Watch this Scientific Journal Video about The In ovo CAM-assay as a Xenograft Model for Sarcoma at JoVE.com

Celler fra SW1353 chondrosarcoma cellelinje blev venligst stillet til rådighed af Prof. Dr. PCW Hogendoorn og Prof. Dr. J. Bovee af Leiden Universitet, Holland. Vi takker J. Mestach og G. Wagemans for fremragende teknisk bistand og G. De Bruyne til den professionelle tegning af overblik over vores protokol.

Se figur 1 for en oversigt. Tumormateriale 1.. Indhentning og forberedelse tumorprøver Til brug for patientens materiale, er godkendelse af Etisk Udvalg er nødvendigt, og informeret samtykke skal indhentes fra patienten. <ol><li> Høst repræsentativt materiale (minimum 1 cm3) ved indgreb, enten en biopsi eller en resektion af en sarkom. Den korrekte site af biopsi er defineret ved hjælp dynamisk kontrast...

<ol>
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(33), e1620 (2009).</li><li>Balke, M., Neumann, A., 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=Morphologic+characterization+of+osteosarcoma+growth+on+the+chick+chorioallantoic+membrane.">Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane.</a> BMC Research Notes. 3, 58 (2010).</li><li>Hendrix, A., Maynard, D., 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=Effect+of+the+secretory+small+GTPase+Rab27B+on+breast+cancer+growth,+invasion,+and+metastasis.">Effect of the secretory small GTPase Rab27B on breast cancer growth, invasion, and metastasis.</a> Journal of the National Cancer Institute. 102, 866-880 (2010).</li><li>Ausprunk, D., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+of+normal+and+neoplastic+tissues+grafted+to+the+chick+chorioallantois:+role+of+host+and+preexisting+graft+vessels.">Vascularization of normal and neoplastic tissues grafted to the chick chorioallantois: role of host and preexisting graft vessels.</a> American Journal of Pathology. 79, 597-618 (1975).</li><li>Lokman, N. 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Tidspunkt for podning og høst Timing dagen for podning blev udført ved hjælp SAOS2 i ECM gel (36 CAMs) og varierede mellem fosterudviklingen dag 5 og 10. Før inkubation dag 9, var CAM ikke konsekvent stor nok til at understøtte ECM gelen vi anvendte. Ved høst, undertiden tumorceller skulle hentes fra dybere CAM, og nogle ECM gelprøver lå løs i æggehviden eller på CAM. På dag 5 og 6, var det svært at undgå at tumorcellerne på hønseembryo, hvilket resu...

Sarkom er en sjælden tumor i bindevæv med en høj dødelighed som følge af behandling modstand 1,2. Fremskridt i patientens overlevelse er hæmmet af deres lave årlige incidens, deres brede mangfoldighed, og det faktum at sarcomaceller rapporteres at være svært at kultur in vitro 3,4. Brugen af dyrkede celler til præklinisk terapi evaluering har vist, at nye, tilsyneladende aktive molekyler in vitro ikke altid afspejler resultater i kliniske omgivelser. Endvidere genom aberrationer afsløret af genekspression arrays er ikke altid korreleret til tumor adfærd karakteristika i den enkelte patient 5-7. For at forsøge at løse disse problemer har skræddersyet medicin fået større betydning, hvilket afspejles i den øgede søgning efter xenograftmodeller 8-12. Et in vivo-assay har den fordel afspejler komplekse samspil mellem Cancer celler og værtens væv miljø i solide tumorer, der er nødvendige for kræft spredning og invasion 13.. I øjeblikket vi undersøge anvendelsen af Chorio-allantoiske Membran assay (CAM-assay) som en reproducerbar xenograft model for sarkom 14,15. Dette assay er almindeligt anvendt til undersøgelse af tumorangiogenese 16,17. I litteraturen har vi imidlertid fundet forskellige protokoller for dette assay, mens andre studier observeret en markant forskel i vækst eller angiogenese i henhold til forskellige protokoller 18,19. I denne artikel vil vi undersøge effekten af ​​varierende betingelser for CAM-analysen på celle adfærd ved hjælp af tumor transplantater, tumorafledte encellede suspensioner og etablerede sarkom cellekulturer.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Cell Line Nucleofector Kit V</td>
			<td>Amaxa</td>
			<td>VCA-1003</td>
		</tr>
		<tr>
			<td>collagenase 2 solution (500 U/ml RPMI 1640)</td>
			<td>Sigma Aldrich</td>
			<td>C6885</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41965-039</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
		</tr>
		<tr>
			<td>Dnase solution</td>
			<td>Sigma Aldrich</td>
			<td>DN25</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>11811031</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E1270</td>
		</tr>
		<tr>
			<td>mouse primary monoclonal antibody Ki67</td>
			<td>Dako Denmark</td>
			<td>MIB-1</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fluka</td>
			<td>D76240</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>20012019</td>
		</tr>
		<tr>
			<td>PBSD</td>
			<td>Invitrogen</td>
			<td>14040083</td>
		</tr>
		<tr>
			<td>peGFP-C1 vector</td>
			<td>Clontech</td>
			<td>632470</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15140163</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Invitrogen</td>
			<td>22409-015</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Invitrogen</td>
			<td>25300054</td>
		</tr>
		<tr>
			<td>Vybrant cell-labeling DiI</td>
			<td>Lifetechnologies</td>
			<td>22885</td>
		</tr>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
		</tr>
		<tr>
			<td>digital color camera</td>
			<td>Leica</td>
			<td>DFC 340 FX</td>
		</tr>
		<tr>
			<td>Digital Egg Incubator</td>
			<td>Auto Elex Co</td>
			<td>R-COM 50</td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>BD Biosciences</td>
			<td>FACSAriaIII</td>
		</tr>
		<tr>
			<td>Gentlemacs C-Tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator User Manual containing h_tumor protocol</td>
			<td>Miltenyi Biotech</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>semipermeable adhesive film (Suprasorb F)</td>
			<td>Lohmann&amp;Rauscher</td>
			<td>20468</td>
		</tr>
		<tr>
			<td>stereo fluorescence microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
		</tr>
		<tr>
			<td>Tissue-Tek Film automated Coverslipper</td>
			<td>Sakura</td>
			<td>6400</td>
		</tr>
		<tr>
			<td>ultraView Universal DAB Detection Kit</td>
			<td>Ventana Medical Systems Inc</td>
			<td>760-500</td>
		</tr>
		<tr>
			<td>Ventana Automated Slide Stainer</td>
			<td>Ventana Medical Systems</td>
			<td>Benchmark XT</td>
		</tr>
	</tbody>
</table>

the in ovo cam-assay as a xenograft model for sarcoma

Sarkom er en meget sjælden sygdom, der er heterogent i naturen, alle hæmmer udviklingen af ​​nye behandlingsformer. Sarkom patienter er ideelle kandidater til skræddersyet medicin efter lagdeling, forklarer den nuværende interesse i at udvikle en reproducerbar og low-cost xenotransplantat model for denne sygdom. Den kyllingechorioallantoinmembranen membranen er en naturlig immundefekt vært stand til at opretholde podede væv og celler uden artsspecifikke restriktioner. Desuden er det let adgang, manipuleres og afbildes ved hjælp af optiske og fluorescens stereomikroskopserien. Histologi tillader endvidere detaljeret analyse af heterotypic cellulære interaktioner. Denne protokol beskriver detaljeret in ovo podning af fosterhinder med friske sarkom-afledte tumorvæv, deres encellede suspensioner og permanente og forbigående fluorescensmærkede etablerede sarkom cellelinjer (Saos-2 og SW1353). Chick overlevelse rottees er op til 75%. Modellen anvendes til at studere graft-(rentabilitet, Ki67 proliferationsindeks, nekrose, infiltration) og vært (fibroblast infiltration, vaskulær indvækst) adfærd. For lokaliseret podning af encellede suspensioner, giver ECM gel betydelige fordele i forhold inaktive indeslutning materialer. Den Ki67 proliferationsindeks er relateret til afstanden af ​​cellerne fra overfladen af ​​modulet og varigheden af ​​anvendelsen på CAM&#39;en, sidstnævnte bestemmelse en tidsramme for tilsætning af terapeutiske produkter.

Evaluering af CAM Tumor podninger bliver klæbende til CAM (figur 2A). Single cellesuspensioner fra patientmateriale ofte vise en tørret, lidt hævet plaque (Figur 2D). Efter udskæring af CAM, markerede rynker af membranen forekommer (figur 2E og 2F). For kommercielle cellelinjer plak bliver mere uigennemsigtige tid, hvilket indikerer celleproliferation. Forskellige cellelinjer i ECM gel har en ty...

Watch this Scientific Journal Video about The In ovo CAM-assay as a Xenograft Model for Sarcoma at JoVE.com

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Den<em&gt; In ovo</em&gt; Chorioallantoide membran (CAM) er podet med friske sarkom-afledte tumorvæv, deres encellede suspensioner og permanente og forbigående fluorescensmærkede etablerede sarkom cellelinjer. Modellen anvendes til at studere graft-(rentabilitet, Ki67 proliferationsindeks, nekrose, infiltration) og vært (fibroblast infiltration, vaskulær indvækst) adfærd.

Den<em&gt; I ovo</em&gt; CAM-assay som et Xenograftmodel til Sarkom

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

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The In ovo CAM-assay as a Xenograft Model for Sarcoma

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The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Etablering af en human myelomatose xenograftmodel i kylling til Studieinformationen tumorvækst, invasion og angiogenese

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

Oliesyre-injektion i svin som en Model for akut respiratorisk Distress syndrom

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Isometrisk kontraktilitet måling af musen mesenterica arterie ved hjælp af Wire blodkar

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

Hornhindens epitel slid med okulær Burr som en Model for hornhinden sårheling

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...