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 ble vennlig levert av professor dr. PCW Hogendoorn og professor Dr. J. Bovee av Leiden University, Nederland. Vi takker J. Mestach og G. Wagemans for utmerket teknisk assistanse, og G. De Bruyne for den profesjonelle tegning av oversikt over protokollen vår.

Se figur 1 for en oversikt. Tumor materialet En. Innhenting og forberede tumorprøver For bruk av pasientmateriale, er godkjenning av Etisk Komité nødvendig, og informert samtykke skal innhentes fra pasienten. <ol><li> Innhøsting representative materiale (minimum 1 cm 3) ved tidspunktet for inngrep, enten en biopsi eller en reseksjon av et sarkom. Den riktige stedet for biopsi er definert ved hjelp av dyn...

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Tid for vaksinasjon og innhøsting Timing dagen for vaksinasjonen ble utført ved hjelp av SAOS2 i ECM gel (36 CAM) og varierte mellom embryonale utvikling dag 5 og 10 år. Før inkubasjon dag 9, var CAM ikke konsekvent stor nok til å støtte ECM gel vi søkte. Ved innhøsting, kreftceller noen ganger måtte hentes fra dypere CAM, og noen ECM gel prøvene ble liggende løs i albumen eller på CAM. På dag 5 og 6, var det vanskelig å unngå å sette kreftceller på...

Sarkom er en sjelden svulst av bindevev med en høy dødelighet som følge av behandling motstand 1,2. Fremgang i pasientens overlevelse er hemmet av deres lave årlig insidens, deres bredt mangfold, og det faktum at sarkom celler er rapportert å være vanskelig å kultur in vitro 3,4. Bruk av dyrkede celler for preklinisk terapi evaluering har avdekket at nye, tilsynelatende aktive molekyler in vitro ikke alltid gjenspeiler resultatene i klinisk setting. Videre genom avvik avdekket av genuttrykk arrays er ikke alltid korrelert til tumor adferdstrekk hos den enkelte pasient 5-7. For å prøve og løse disse problemene, har personlig medisin fått stadig større betydning, noe som gjenspeiles i økt søk etter xenograft modeller 8-12. Et in vivo-forsøk har fordelen av å reflektere det komplekse samspill mellom cancer celler og verten vev miljø i solide svulster, er nødvendig for kreft spredning og invasjon 13. For tiden vi studerer bruken av Chorio-Allantoic Membrane assay (CAM-analyse) som en reproduserbar xenograft modell for sarkom 14,15. Denne analysen er mye brukt for studiet av tumor angiogenese 16,17. I litteraturen, har vi imidlertid funnet forskjellige protokoller for denne analysen, mens andre studier observert en markert forskjell i veksten eller angiogenese i henhold til forskjellige protokoller 18,19. I denne artikkelen undersøker vi effekten av varierende forhold i CAM-analysen på celle atferd med svulst grafts, tumor-avledet encellede suspensjoner og etablerte 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 sjelden sykdom som er heterogen i naturen, alle hindre utviklingen av nye behandlingsformer. Sarkom pasienter er ideelle kandidater for personlig medisin etter lagdeling, forklarer den nåværende interesse i å utvikle en reproduserbar og rimelig xenotransplant modell for denne sykdommen. Den dama chorioallantoic membranen er en naturlig immunodeficient vert i stand til å opprettholde podet vev og celler uten artsspesifikke restriksjoner. I tillegg er det lett tilgjengelig, manipulert og avbildes ved hjelp av optisk og fluorescens stereomikroskopi. Histologi tillater ytterligere detaljert analyse av heterotypic cellulære interaksjoner. Denne protokollen beskriver i detalj i ovo pode av chorioallantoic membran med ferske sarkom-avledet tumorvev, deres encellede suspensjoner, og permanente og forbigående fluorescensmerkede etablerte sarkom cellelinjer (SAOS-2 og SW1353). Den dama overlevelse rottees er opptil 75%. Modellen brukes til å studere pode-(levedyktighet, Ki67 spredning indeks, nekrose, infiltrasjon) og vert (fibroblast infiltrasjon, vaskulær ingrowth) atferd. For lokaliserte poding av encellede suspensjoner, gir ECM gel betydelige fordeler fremfor inerte containment materialer. Den Ki67-indeks spredning er relatert til avstanden av cellene fra overflaten av CAM og varigheten av programmet på CAM, idet sistnevnte bestemme en tidsperiode for tilførsel av terapeutiske produkter.

Evaluering av CAM Tumor grafts bli tilhenger til CAM (Figur 2A). Encellede suspensjoner fra pasientmateriale ofte vise en tørket, litt hevet plakk (figur 2D). Etter fjerning av CAM, markerte rynker av membranen skjer (Tall 2E og 2F). For den kommersielle cellelinjer plakk blir mer ugjennomsiktig i tid, noe som indikerer celleproliferasjon. Forskjellige cellelinjer i ECM gel har en tydelig vekst mø...

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; I ovo</em&gt; Chorioallantoic membran (CAM) er podet med ferske sarkom-deriverte tumorvev, deres encellede suspensjoner, og permanente og forbigående fluorescensmerkede etablerte sarkom cellelinjer. Modellen brukes til å studere pode-(levedyktighet, Ki67 spredning indeks, nekrose, infiltrasjon) og vert (fibroblast infiltrasjon, vaskulær ingrowth) atferd.

Den<em&gt; I ovo</em&gt; CAM-analysen som en Xenograftmodel for 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 av et menneske multippelt myelom Xenotransplantat Model i Chicken å studere tumorvekst, invasjon og Angiogenesis

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

Oljesyre-injeksjon i griser som en modell for akutt Respiratory 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 Contractility måling av musen hvem arterien bruker Wire Myography

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

Hornhinnen Epithelial slitasje med okulær Burr som modell for hornhinnen sårtilheling

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 ...