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.

Il protocollo che segue è effettuata secondo le linee guida stabilite dal Dipartimento di Medicina di Laboratorio animali presso la University of North Carolina a Chapel Hill. 1. Preparare il seguente materiale e reagenti prima di iniziare <ol><li> Preparare supporti CE, completandola 400 ml glucosio basso (1 g / l D-glucosio o LG) Dulbecco Modified mezzo di Eagle (DMEM) con 50 ml di siero fetale bovino inattivato al calore, 50 ml di Nu-Siero IV, 5 ml di antibiotico-antimicotico, e la hFGF, VEGF, hEGF, R3-IGF-1, e componenti eparina dal kit commerciale. </li><li> Preparare 500 ml FACS tampone (0,5% BSA e 2 mM EDTA in PBS); filtrare attraverso una sterile 0,22 micron coppa del filtro. </li><li> Sterilizzare o disinfettare dissezione bordo. </li><li> Sterilizzare perni dissezione, forbici chirurgiche e dissettori. </li></ol> 2. Isolamento CE (1 ° giorno, ~ 5 ore) <ol><li> Euthanize mouse con anidride carbonica o altri metodi di spirito compatibileh la cura e l&#39;uso degli animali Comitato Istituzionale (IACUC) politiche. Nota: Più tumori mammari di dimensioni variabili (5 - 15 mm di diametro) possono sviluppare in un singolo mouse geneticamente modificati come C3-Tag, assicurarsi di raccogliere tutti loro per l&#39;isolamento TEC. Se si utilizza tumori che sono ortotopicamente innestato nei topi, piscina due o tre 1 centimetro 3 tumori per un singolo isolamento CE. Qui usiamo tumori mammari come dimostrazione, ma il protocollo può essere modificato per altri tipi di tumori. </li><li> Disinfettare topo nebulizzare o passare la parte ventrale mouse con ampie quantità di 75% v / v di etanolo. </li><li> Resecare tumori con un paio di forbici e dissettori utilizzando tecniche asettiche in una cappa sterile o in un banco pulito. <ol><li> Stendi e pin le membra del mouse su una scheda di dissezione. Effettuare una incisione ventrale linea mediana con le forbici senza aprire il peritoneo. Sezionare lateralmente tra la pelle ed il peritoneo verso le ghiandole mammarie in cui si trovano i tumori.Non aprire il peritoneo. </li><li> Accise solo tessuto tumorale dalle ghiandole mammarie, lasciando fuori normali margini mammarie. Rifilare con cura off tessuti non tumorali, come la pelle ei muscoli, e il luogo tumori sezionato in una provetta contenente 30 ml di LG-DMEM su ghiaccio. </li></ol></li><li> Portare campioni di tumore alla cappa coltura di tessuti; lavare i tessuti con sterile LG-DMEM 1 - 2 volte. </li><li> Trasferire i tumori del tubo conico di una capsula di Petri tessuto-cultura sterili, aggiungere 2 ml di LG-DMEM nel piatto, e tritare con un paio di forbici sterili in pezzi &lt;5 mm. </li><li> Aggiungere 5 ml di collagenasi (magazzino = 2 mg / ml in soluzione salina bilanciata di Hank, seguito HBSS), 1 ml di dispasi (magazzino = 2,5 U / ml in HBSS) e 75 ml di deossiribonucleasi (magazzino = 1 mg / ml in PBS ) nella capsula di Petri. Il volume totale è ora ~ 10 ml. </li><li> Trasferire il mix collagenasi / tessuto dal capsula di Petri di un tubo di tessuto-dissociatore ed eseguire su un dissociatore tessuto per 60 secondi per due volte (pre-impostato di dissociazione prOgram sul dissociatore: 1.270 giri totali per corsa). Incubare con la luce agitando su un agitatore per 75 min a 37 ° C. </li><li> Filtro digerito tessuto attraverso un colino cella 100 micron su una provetta conica da 50 ml. Filtro Sciacquare con 5 ml di tampone FACS per lavare tutte le cellule rimanenti. Spin a 280 xg per 5 minuti ed aspirare accuratamente il surnatante senza disturbare il pellet. </li><li> Diluire 1 ml di brodo RBC tampone di lisi (10x) in 9 ml di acqua sterile. Lisare globuli rossi con 10 ml di tampone di lisi (1x), e subito girare 5 min a 280 x g. Nota: Questo passaggio può essere saltato se po &#39;di sangue è visibile. </li><li> Risospendere in 10 ml di tampone FACS. Miscelare 10 ml di sospensione cellulare con 10 ml di trypan blu, e contare le cellule vive con un emocitometro. </li><li> Risospendere le cellule a ~ 10 7 cellule / 100 ml. Aggiungere 10 ml di FcR blocco per 100 ml di sospensione cellulare e incubare in ghiaccio per 10 min. </li><li> Aggiungere anticorpi CD31 ratto anti topo PE coniugato secondo. alla Tabella 1 Incubare in ghiaccio per 10-15 minuti e tubo flick di tanto in tanto. </li><li> Aggiungere 10 ml di tampone FACS al tubo e girare a 280 xg per 5 minuti. Rimuovere con attenzione il surnatante, e lavare il pellet cellulare nuovo con 5 ml di tampone FACS. Centrifuga a pellet cellule. Aspirare il surnatante senza pellet inquietante. </li><li> Aggiungere FACS tampone e anti-PE microsfere secondo la Tabella 2 Incubare in ghiaccio per 10 -. 15 min. Flick tubo di tanto in tanto. </li><li> Aggiungere 10 ml di FACS tampone e di spin campioni a 280 xg per 5 minuti; Risciacquare una volta con 5 ml di tampone FACS e centrifugare nuovamente. Aspirare il surnatante senza disturbare pellet. </li><li> Portare il volume a 300 ml in tampone FACS. Spin attraverso 35 micron cellula-filtro ricoperto tubo 5 min a 280 x g. Utilizzare 2 tubi e un volume maggiore, se necessario. </li><li> Impostare multistand magnetica e colonne magnetici in cappuccio, collegare una colonna al separatore ed equilibrare la colonna con 2 ml di tampone FACS. </li><li> Aspirate il surnatante e risospendere il pellet cellulare in 0,5 - 1 ml di tampone FACS. </li><li> Passare la sospensione cellulare attraverso la colonna magnetica equilibrata. </li><li> Lavare la colonna tre volte con 2 ml di tampone FACS, e raccogliere flow-through (FT) in un tubo di 15 ml (frazione FT). </li><li> Prendere colonna fuori il separatore ed eluire con 2 ml di tampone FACS in un altro tubo 15 ml (frazione di eluato). Utilizzare stantuffo per garantire tutte le cellule sono fuori dalla colonna. Ripetere la eluizione altre due volte con 2 ml di tampone FACS ogni volta. </li><li> Spin l&#39;eluato a 280 xg per 5 min. </li><li> Rimuovere il surnatante e risospendere il pellet in 10 ml di media CE. </li><li> Altrettanto dividere la frazione di eluato (~ 6 ml) in 10 piatti cm rivestiti di gelatina. Per tre 1 cm 3 tumori, le cellule piastra eluita in almeno quattro piatti. Cellule alternativa, piatto eluito a diverse concentrazioni in più lastre (ad es., Seminare 0,5 ml, 1 ml, 1,5 ml e 3 ml di eluato in quattro piatti) per garantire che unat almeno una piastra è scarsamente seminato con le cellule eluite. Verificare che la confluenza delle cellule attaccate è ~ 1% il giorno dopo (cioè, circa 1,0 - 2,0 x 10 5 cellule allegate). Nota: le cellule devono essere placcato sparso in modo che le colonie CE possono formarsi senza essere contaminato da altri tipi di cellule. </li><li> Piastra frazione FT in un piatto 10 centimetri di lasciare le cellule recuperare O / N. Controllare che il piatto è 80-100% confluenti il ​​giorno successivo. Congelare le cellule (-80 ° C) a 250 supporti cellulari congelamento microlitri in un cryotube e conservarli in azoto liquido il giorno successivo. Nota: frazione FT può essere utilizzato per l&#39;isolamento delle cellule tumorali in una fase successiva e / o come controllo negativo per l&#39;analisi di espressione genica CE dei cloni isolati CE. </li><li> Modificare i media ogni 2 - 3 giorni. Colonies cominciano a formarsi dopo 7 - 10 giorni. Colonie piccole CE possono essere individuati prima giornata 3. Mark le colonie con un pennarello fine punta sul fondo del piatto. </li><li> Raschiare non specifico cells circondano le colonie identificate con una sterile, punta Pippet 200 microlitri. </li></ol> 3. Colony selezione Anelli Usare Clonazione <ol><li> Modificare i media ogni 2 - 3 giorni. Purezza CE può essere controllato da LDL (DII-Ac-LDL), oltre a circa 5-7 giorni. Aggiungere 50 ml di LDL per 10 ml di mezzo CE e incubare per 3-4 ore prima di controllare le cellule al microscopio a fluorescenza. Nota: Multiple LDL + cloni CE possono essere osservati a ~ 7 giorni. </li><li> Inizia la raccolta colonie CE quando raggiungono diametri di 3 - 5 mm di dimensione. (Scegliere grandi colonie che sono pieni di piccoli LDL + cellule per ottenere i migliori risultati.) </li><li> Prima della raccolta CE con anelli di clonazione, pre-coat alcune 6 pozzetti con 0,5% di gelatina. Aspirare gelatina, aggiungere 2 ml di media CE per ogni bene, e tenere le piastre in un incubatore fino al momento. </li><li> Raschiare non CE sui bordi delle colonie per assicurarsi che non altri tipi di cellule saranno intrappolati all&#39;interno dell&#39;anello clonazione. </li><li> Utilizzando unmicroscopio a contrasto di fase (4X o 10X obiettivo), contorno con un pennarello fine-tip sul fondo del piatto cultura delle aree contenenti colonie CE. </li><li> Lavare la piastra con 10 ml di PBS e lasciare uno strato molto sottile di PBS nella piastra durante l&#39;aspirazione. (Importante: una piccola quantità (~ 0,5 ml) di PBS manterrà cellule vive durante la procedura di clonazione-ring, anche, tessuto adesivo ha bisogno di acqua per legare.) </li><li> Scegliere un anello di clonazione di dimensioni adeguate. Utilizzare un paio di dissettore a prendere un anello, e con una punta di pipetta 10 microlitri uniformemente applicare una piccola quantità di tessuto adesivo sull&#39;anello clonazione. Nota: utilizzare solo minima quantità (~ 0,2 microlitri per un piccolo anello) di tessuto adesivo su anelli di clonazione, e assicurarsi che il tessuto adesivo è distribuito in modo uniforme intorno alla superficie di fondo per garantire una buona tenuta. Tessuto adesivo eccessivo produce calore e forma film che possono uccidere le cellule. </li><li> Posizionare l&#39;anello di clonazione sopra la colonia CE. Premere delicatamente verso il basso l&#39;anello di clonazione per incollare i ring sul piatto. Assicurarsi che le colonie non sono asciugato prima di incollare l&#39;anello. </li><li> Immediatamente pipettare 25 ml di soluzione di distacco delle cellule enzimatica sul ring clonazione e incubare ~ 1 min o fino a quando le cellule sono vagamente collegati. </li><li> Cellule pipetta sul ring clonazione goccia a goccia in un pozzetto di un 6-pozzetti contenenti supporti CE pre-riscaldato. (Importante: Non agitare la piastra per disperdere le cellule, CE preferiscono crescere in ammassi stretti.) Lavare l&#39;anello di clonazione con 50 - 100 supporti CE microlitri di raccogliere il maggior numero di cellule possibile e trasferire tutti i lavaggi nello stesso 6-bene . </li><li> Se alcune colonie nel piatto 10 centimetri sono troppo piccoli per raccogliere, aggiungere 10 ml di mezzi freschi, lasciare che le colonie crescono per qualche giorno in più, e ripetere la procedura di clonazione anello. </li><li> Crescere colonie raccolte in 6 pozzetti fino a 80-100% confluenti, e il trasferimento di cellule a 3 pozzetti di una 6-pozzetti, prima di espandersi in un 10 centimetri piatto di coltura di tessuti. Raschiare le cellule contaminanti. Ripetereun altro giro di procedura anello clonazione (i passaggi 3,5-3,10) se necessario. Mantenere le cellule relativamente confluenti (~ 60 - 70%) durante l&#39;espansione. CE può smettere di crescere se placcato troppo scarsa. </li><li> Caratterizzare CE da FACS, la colorazione, la PCR, ecc Si noti che Dil-Ac-LDL è fluorescente e può interferire con PE o altri anticorpi fluorescenti per FACS. </li></ol>

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Med. 6 (227), 227ra34-227ra34 (2014).</li><li>Garcia, J., 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=Tie1+deficiency+induces+endothelial-mesenchymal+transition.">Tie1 deficiency induces endothelial-mesenchymal transition.</a> EMBO Rep. 13 (5), 431-439 (2012).</li><li>Xiao, L., 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=Tumor+endothelial+cells+with+distinct+patterns+of+TGF&#946;-driven+endothelial-to-mesenchymal+transition.">Tumor endothelial cells with distinct patterns of TGF&#946;-driven endothelial-to-mesenchymal transition.</a> Cancer Res. 75 (7), 1244-1254 (2015).</li><li>Kusumbe, A. P., Ramasamy, S. K., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+angiogenesis+and+osteogenesis+by+a+specific+vessel+subtype+in+bone.">Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.</a> Nature. 507 (7492), 323-328 (2014).</li><li>Wang, L., 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=Identification+of+a+clonally+expanding+haematopoietic+compartment+in+bone+marrow.">Identification of a clonally expanding haematopoietic compartment in bone marrow.</a> EMBO J. 32 (2), 219-230 (2012).</li><li>Sawamiphak, S., 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=Ephrin-B2+regulates+VEGFR2+function+in+developmental+and+tumour+angiogenesis.">Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.</a> Nature. 465 (7297), 487-491 (2010).</li><li>Chi, J. -. T., 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=Endothelial+cell+diversity+revealed+by+global+expression+profiling.">Endothelial cell diversity revealed by global expression profiling.</a> Proc. Natl. Acad. Sci. 100 (19), 10623-10628 (2003).</li><li>Nolan, D. J., 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=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev. Cell. 26 (2), 204-219 (2013).</li><li>Ingram, D. 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=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105 (7), 2783-2786 (2005).</li></ol>

The authors have nothing to disclose.

A causa delle difficoltà di ottenere colture TEC primaria pure, molti studi in vitro TCE sostituto con le linee disponibili in commercio CE o primaria CE, come vena ombelicale umano CE (HUVEC) 13. Tuttavia, queste popolazioni CE dai tessuti normali possono servire solo come un proxy per TEC che si differenziano nettamente dalle loro controparti normali. Ad esempio, TEC sono fenotipicamente e funzionalmente anormale in vivo e alcune di queste anomalie può essere trasmissibile in vitro</...

Le cellule endoteliali (EC) sono essenziali durante lo sviluppo di tumori solidi. Da inizio dello switch angiogenico nei tumori dormienti per la diffusione e la semina delle metastasi a siti distanti, CE formano i condotti che forniscono sangue, ossigeno e sostanze nutritive per sostenere la crescita del tumore 1. Come recentemente suggerito, CE hanno anche funzioni di perfusione indipendente e formano una nicchia che supporta la crescita delle cellule staminali del cancro e di altre cellule stromali del tumore 2-5. Così, altamente purificato CE (TCE) per la cultura in vitro tumore-specifica permette di studi funzionali di routine che faranno luce sui meccanismi molecolari che mediano l&#39;angiogenesi tumorale e diafonia con le cellule tumorali. CE sono altamente specializzata a seconda del tessuto di origine 6. A causa della natura eterogenea dei diversi tipi di tumore e il microambiente tumorale, TEC può anche visualizzare le caratteristiche uniche che riflettono un tumore-specifica specializzazione of il sistema vascolare. Ad esempio, vi è la variabilità sorprendente nelle firme di espressione genica nel TEC isolate da diversi tipi o categorie di tumori 7,8. Tuttavia, frequenti co-purificazione di non CE, in particolare fibroblasti associati al tumore e le cellule tumorali, con TEC può confondere le analisi di espressione su scala genomica. Questi tipi di cellule indesiderate sono particolarmente problematico in studi che si basano su a lungo termine in espansione in vitro di culture TEC. Descritto qui è un metodo ad alta fedeltà che produce costantemente le colture pure CE da tumori e di altri tessuti. A seguito di colonna immunomagnetica arricchimento delle frazioni e la rimozione di co-purificato non CE CE, un passo clonazione anello aggiuntivo per catturare colonie puri CE viene utilizzato 9. Ogni colonia può essere espansa in coltura per più passaggi, senza l&#39;emergere di contaminazione non CE. Questo metodo produce anche più cloni CE da una singola procedura di isolamento, che è ideale per lo studio di endoeterogeneità thelial. Inoltre, è dimostrato che Cdh5 cre: / l topi ZsGreen l / s giornalista sono uno strumento prezioso per la generazione EC &quot;destino-mapped&quot; e indelebilmente marcato che mantengono ZsGreen fluorescenza nella cultura 10. Con piccole modifiche al protocollo, questo metodo dovrebbe essere adattabile a diversi tipi di tumore o tessuti normali.

<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

Appena isolate tumore-specifici cellule endoteliali (TEC) possono essere usati per esplorare i meccanismi molecolari di angiogenesi tumorale e servire come un modello in vitro per lo sviluppo di nuovi inibitori dell&#39;angiogenesi per il cancro. Tuttavia, a lungo termine in espansione in vitro di cellule endoteliali murine (EC) è difficile a causa di deriva fenotipica nella cultura (passaggio endoteliale-a-mesenchimale) e la contaminazione con i non-CE. Ciò è particolarmente vero per TEC, che sono prontamente outcompeted da fibroblasti co-purificate o cellule tumorali in coltura. Qui, un metodo di isolamento ad alta fedeltà che sfrutta arricchimento immunomagnetica accoppiato con selezione delle colonie e in espansione in vitro è descritto. Questo approccio genera frazioni pure CE che sono completamente libero di contaminare cellule stromali o tumorali. E &#39;inoltre dimostrato che lignaggio tracciato Cdh5 cre: / l topi reporter ZsGreen l / s, utilizzati con il protocollo qui descritto, sono un valido strumento per verificare cellularepurezza isolate colonie CE di questi topi mostrano durevole e brillante fluorescenza ZsGreen nella cultura.

CE rappresentano soltanto una frazione minore della popolazione cellulare totale nella maggior parte dei tessuti adulti 11. È quindi importante digerire completamente il tessuto raccolto in una cella singola sospensione che assicura il rilascio massimo CE da matrice extracellulare (ECM) e dei tessuti connettivi. Nella nostra esperienza, la selezione immunomagnetica CD31-mediata prevede solo arricchite, ma non puri frazioni CE; Pertanto, un altro passo fondamentale è la rimozione fisica di co-purificata non ...

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.

Isolamento e cultura espansione delle cellule endoteliali tumore-specifici

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

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

Research

JoVE Journal

Medicine

11.1K Views.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 

Video: Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Ricerca

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

Isolamento, Cultura e Imaging di Human Fetal pancreatici gruppi di cellule

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Isolamento, Crioconservazione e Cultura umani Cellule epiteliali Amnion per le applicazioni cliniche

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolamento di cellule umane endoteliali linfatico da Multi-parametro fluorescenza-attivato cell sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Isolamento e coltura di cellule endoteliali primarie dalle arterie e vene Canine

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

L&#39;isolamento di cellule progenitrici endoteliali da volontari sani e il loro potenziale migratori influenzato dal siero campioni dopo cardiochirurgia

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

preparazione e<em&gt; In Vitro</em&gt; Caratterizzazione di magnetizzato miR-modificato cellule endoteliali

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

Valutazione delle proprietà anticoagulanti e anti-infiammatori delle cellule endoteliali mediante coltura cellulare 3D e Non intero anticoagulato

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Squilibri muscolari: Test e formazione forza eccentrica funzionale del tendine del ginocchio nelle popolazioni di atletiche

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...