Name: isolation and culture expansion of tumor-specific endothelial cells
Uploaded: 2015-10-14T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

Het volgende protocol wordt uitgevoerd op basis van door het ministerie van Laboratory Animal Geneeskunde aan de Universiteit van North Carolina in Chapel Hill richtlijnen. 1. Bereid het volgende materiaal en reagentia Voordat u begint <ol><li> Bereid EC media door aanvulling 400 ml lage glucose (1 g / l D-glucose of LG) Dulbecco&#39;s gemodificeerd Eagle&#39;s medium (DMEM) met 50 ml hitte-geïnactiveerd foetaal runderserum, 50 ml Nu-Serum IV, 5 ml antibioticum-antimycoticum, en hFGF, VEG...

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Vanwege de moeilijkheden bij het ​​verkrijgen van zuivere primaire kweken TEC, veel in vitro studies TEC vervanging met commercieel verkrijgbaar EC lijnen of primaire EC zoals humane navelstreng EC (HUVEC) 13. Echter, kunnen deze EC populatie van normale weefsels alleen dienen als een proxy voor de TEC, die sterk afwijken van hun normale tegenhangers. Zo TEC fenotypisch en functioneel abnormale in vivo en sommige van deze afwijkingen kunnen overdraagbare in vitro 1...

Endotheelcellen (EC) zijn essentieel tijdens de groei van grote tumoren. Vanaf de start van de angiogene switch in slapende tumoren verspreiding en het zaaien van metastasen op afgelegen locaties, EG vormen de leidingen die het bloed, zuurstof en voedingsstoffen om 1 tumorgroei ondersteunen. Zoals onlangs gesuggereerd, EC ook perfusie-onafhankelijke functies en vormen een nis die de groei van kanker stamcellen en andere tumorale stromacellen 05/02 ondersteunt. Zo sterk gezuiverd tumor-specifieke EG (TEC) voor in vitro cultuur zorgt voor routine functionele studies die licht zal werpen op nieuwe moleculaire mechanismen bemiddelen tumor angiogenese en overspraak met tumorcellen. EC zijn zeer gespecialiseerde afhankelijk van het weefsel van oorsprong 6. Vanwege de heterogeniteit van verschillende tumortypes en de tumor micro-omgeving, kan TEC ook unieke eigenschappen die een tumor-specifiek specialisatie o weerspiegelen tonenf het vaatstelsel. Zo is er opvallende variatie in genexpressie handtekeningen TEC geïsoleerd van verschillende types of klassen van tumoren 7,8. Echter, kunnen frequente co-zuivering van niet-EG, in het bijzonder tumor-geassocieerde fibroblasten en tumorcellen, met TEC verwarren genoom-wijde expressie analyses. Deze ongewenste celsoorten zijn bijzonder problematisch studies die afhankelijk lange termijn in vitro expansie van TEC culturen. Hier beschreven is een high-fidelity methode die consistent produceert zuivere cultures van EC tumoren en andere weefsels. Volgende kolom immunomagnetische verrijking van EG-fracties en verwijdering van co-gezuiverde niet-EG, een extra klonen-ring stap om pure EG kolonies wordt gebruikt 9 vast te leggen. Elke kolonie kan in de cultuur voor meerdere passages uit te breiden zonder de opkomst van verontreinigende niet-EG. Deze werkwijze levert ook meerdere EC klonen uit een isolatiewerkwijze, die ideaal is voor de studie van endothelial heterogeniteit. Daarnaast wordt aangetoond dat Cdh5 cre: ZsGreen l / s / l reporter muizen zijn een waardevol instrument voor het genereren van &quot;-lot in kaart gebracht&quot; en onuitwisbaar gemarkeerde EG, die ZsGreen fluorescentie in cultuur 10 te handhaven. Met kleine aanpassingen aan het protocol, moet deze methode aan te passen aan verschillende soorten tumoren of normale weefsels zijn.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s medium (1 g/L D-glucose) (LG-DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Bullet Kit&#160;</td>
			<td>Lonza</td>
			<td>CC4176</td>
			<td>Not all components used</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (Hyclone)</td>
			<td>Thermo Scientific</td>
			<td>SH30071.03</td>
			<td>Heat inactivated at 56&#176;C for 30 min</td>
		</tr>
		<tr>
			<td>Nu-Serum IV</td>
			<td>Corning</td>
			<td>CB-51004</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBBS)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &#956;m filter</td>
		</tr>
		<tr>
			<td>75% v/v ethanol for disinfection</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE microbeads&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) fraction V, 7.5%</td>
			<td>Gibco</td>
			<td>15260-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell freezing media (Bambanker)</td>
			<td>Wako Chemicals</td>
			<td>302-14681</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II&#160;&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004176</td>
			<td>Make stock concentration 2 mg/ml in HBSS</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (DNase)</td>
			<td>Worthington Biochemical</td>
			<td>LS002004</td>
			<td>Make stock concentration 1 mg/mL in PBS</td>
		</tr>
		<tr>
			<td>Dil-Ac-LDL</td>
			<td>Biomedical Technologies</td>
			<td>BT-902</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M, pH 8.0</td>
			<td>Cellgro</td>
			<td>46-034-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic cell detachment solution (Accutase)</td>
			<td>Sigma-Aldrich</td>
			<td>A6964-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin, 2 % in water, tissue culture grade</td>
			<td>Sigma-Aldrich</td>
			<td>G1393-100ML</td>
			<td>Dilute in PBS to make 0.5 % gelatin solution</td>
		</tr>
		<tr>
			<td>Mouse FcR Blocking Reagent&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral protease (Dispase)</td>
			<td>Worthington Biochemical</td>
			<td>LS02104</td>
			<td>Make stock concentration 2.5 U/mL in HBSS</td>
		</tr>
		<tr>
			<td>PE-rat anti-mouse CD31 antibody</td>
			<td>BD Pharmingen</td>
			<td>553373</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer (BD Pharm Lyse)</td>
			<td>BD Pharmingen</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4 %&#160;</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mm tissue culture dishes</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes (sterile)</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes (sterile)&#160;</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-dissociator tubes (gentleMACS) C tubes)&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator&#160; (MidiMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 &#956;m&#160;</td>
			<td>Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning rings (assorted sizes)</td>
			<td>Bel-Art Products</td>
			<td>378470000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting board</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize or disinfect with 75% v/v ethanol before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting forceps and scissors</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting pins 2&#34;</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>FACS tubes with 35 &#956;m filter cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter cup (Stericup, 0.22 &#956;m)</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tip marker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Multistand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Or a centrifuge with similar capacity for 15 mL and 50 mL conical tube centrifugation</td>
		</tr>
		<tr>
			<td>Tissue culture hood</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue dissociator (gentleMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td>Preset program &#34;m_impTumor_01&#34; used for tissue dissociation&#160;</td>
		</tr>
		<tr>
			<td>Liquid nitrogen freezer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate or rotary shaker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast light microscope</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture expansion of tumor-specific endothelial cells

Vers geïsoleerde tumorspecifieke endotheelcellen (TEC) kan worden gebruikt om de moleculaire mechanismen van tumorangiogenese staand en dienen als een in vitro model voor de ontwikkeling van nieuwe angiogeneseremmers voor kanker. Echter, de lange-termijn in vitro expansie van muis endotheelcellen (EC) is een uitdaging vanwege fenotypische drift in kweek (endotheel-to-mesenchymale overgang) en verontreiniging met niet-EC. Dit geldt vooral voor TEC die gemakkelijk worden weggeconcurreerd door co-gezuiverd fibroblasten of tumorcellen in kweek. Hier wordt een high fidelity isolatiewerkwijze die gebruik maken van immunomagnetische verrijking gekoppeld kolonieselectie en in vitro expansie neemt beschreven. Deze aanpak levert pure EG fracties die volledig vrij zijn van verontreinigende stromale of tumorcellen. Ook is aangetoond dat lineage getraceerd Cdh5 cre: ZsGreen l / s / l reporter muizen gebruikt met de hierin beschreven protocol, een waardevol hulpmiddel om cellen te controlerenzuiverheid van de geïsoleerde EG kolonies van deze muizen tonen duurzaam en briljante ZsGreen fluorescentie in cultuur.

EG vormen slechts een kleine fractie van de totale celpopulatie in de meeste volwassen weefsels 11. Het is daarom belangrijk om volledig te verteren het geoogste weefsel in een enkele-celsuspensie die de maximale afgifte van EG extracellulaire matrix (ECM) en bindweefsel zorgt. In onze ervaring, CD31-gemedieerde immunomagnetische selectie biedt alleen verrijkt, maar niet zuiver EG fracties; dus een cruciale stap is fysische verwijdering van co-gezuiverde niet-EC en selectie / expansie van EG kolonies gebruik ...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Isolatie en Cultuur Uitbreiding van Tumor-specifieke endotheelcellen

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Research

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Medicine

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Isolatie, karakterisering en vergelijkende differentiatie van humane Dental Pulp stamcellen uit permanente tanden met behulp van twee verschillende methoden

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