Name: establishment of a clinic-based biorepository
Uploaded: 2017-05-29T14:00:00
Description: Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Questo lavoro è stato sostenuto dai fondi del Midwestern University College of Health Sciences per la Facilitazione della Ricerca, concesso a EEH e all&#39;Ufficio Midwestern Office of Research e ai Programmi Sponsorizzati Intramural Grant, assegnato a KJL. Ulteriori sostegno è stato fornito da Affiliated Laboratories e Dermatologia Affiliated. Ringraziamo Sarah Potekhen, Jamie Barto, Stefani Fawks, Cody Jording, Stacie Schimke, Heather Kissel e Ali Zaidi per la loro assistenza tecnica.

Tutta la sperimentazione sui campioni di tessuto umano è stata approvata da Western IRB (Protocollo WIRB # 20142461). Il singolo criterio per la raccolta dei tessuti per queste procedure è una diagnosi bioscopica di SCC. Nessun criterio di esclusione è stato descritto nel protocollo originale IRB, ma non sono stati utilizzati campioni di pazienti con una nota patologia trasmessa dal sangue. Il consenso informato è stato ottenuto prima della raccolta del tessuto cutaneo, del sangue e della saliva. La commissione di r...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+to+assess+tissue+quality.">Tools to assess tissue quality.</a> Clin Biochem. 47, 280-287 (2014).</li><li>Vaught, 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=An+NCI+perspective+on+creating+sustainable+biospecimen+resources.">An NCI perspective on creating sustainable biospecimen resources.</a> J Natl Cancer Inst Monogr. 2011, 1-7 (2011).</li><li>Fu, T., Aasi, S. Z., Hollmig, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+high-risk+squamous+cell+carcinoma+of+the+skin.">Management of high-risk squamous cell carcinoma of the skin.</a> Curr Treat Options Oncol. 17, (2016).</li><li>Rasmussen, C., Thomas-Virnig, C., Allen-Hoffmann, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+human+epidermal+keratinocyte+cell+culture.">Classical human epidermal keratinocyte cell culture.</a> Methods Mol Biol. 945, 161-175 (2013).</li><li>Purdie, K. J., Pourreyron, C., South, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+culture:+Methods+and+protocols.">Cancer cell culture: Methods and protocols.</a> , 151-159 (2011).</li><li>Berglund, S. R., Schwietert, C. W., Jones, A. A., Stern, R. L., Lehman, J., Goldberg, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+methodology+for+sequential+extraction+of+RNA+and+protein+from+small+human+skin+biopsies.">Optimized methodology for sequential extraction of RNA and protein from small human skin biopsies.</a> J Invest Dermatol. 127, 349-353 (2007).</li><li>Schroeder, 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=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7, 1-14 (2006).</li><li>Cooper, H. 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=Expression+and+glycosylation+of+MUC1+in+epidermolysis+bullosa-associated+and+sporadic+cutaneous+squamous+cell+carcinomas.">Expression and glycosylation of MUC1 in epidermolysis bullosa-associated and sporadic cutaneous squamous cell carcinomas.</a> Br J Dermatol. 151, 540-545 (2004).</li><li>Riihila, P. M., 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=Complement+factor+H:+a+biomarker+for+progression+of+cutaneous+squamous+cell+carcinoma.">Complement factor H: a biomarker for progression of cutaneous squamous cell carcinoma.</a> J Invest Dermatol. 134, 498-506 (2014).</li><li>Ha Lan, T. 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=Expression+of+the+p40+isoform+of+p63+has+high+specificity+for+cutaneous+sarcomatoid+squamous+cell+carcinoma.">Expression of the p40 isoform of p63 has high specificity for cutaneous sarcomatoid squamous cell carcinoma.</a> J Cutan Pathol. 41, 831-838 (2014).</li><li>Alomari, A. K., Glusac, E. J., McNiff, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p40+is+a+more+specific+marker+than+p63+for+cutaneous+poorly+differentiated+squamous+cell+carcinoma.">p40 is a more specific marker than p63 for cutaneous poorly differentiated squamous cell carcinoma.</a> J Cutan Pathol. 41, 839-845 (2014).</li><li>Qiao, B., Johnson, N. W., Gao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transition+in+oral+squamous+cell+carcinoma+triggered+by+transforming+growth+factor-beta1+is+Snail+family-dependent+and+correlates+with+matrix+metalloproteinase-2+and+-9+expressions.">Epithelial-mesenchymal transition in oral squamous cell carcinoma triggered by transforming growth factor-beta1 is Snail family-dependent and correlates with matrix metalloproteinase-2 and -9 expressions.</a> Int J Oncol. 37, 663-668 (2010).</li><li>Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+biospecimen+research.">Developments in biospecimen research.</a> Br Med Bull. 114, 29-38 (2015).</li></ol>

Alla conoscenza dell&#39;autore, questo protocollo è il primo del suo genere che si concentra sull&#39;approvazione clinica di campioni di tessuto cutaneo in un approccio economico e veloce. I pazienti sottoposti a microchirurgia di Mohs sono in genere pianificati durante specifici blocchi di tempo e la raccolta è limitata a questi periodi. La raccolta di campioni comporta l&#39;impegno dell&#39;assistente medico coinvolto nella cura del paziente, del histotechnolog Mohs che elabora i campioni tumorali e un membro del...

La ricerca sul cancro e sul biomarker si basa su una fornitura di campioni di tessuti umani di qualità e l&#39;offerta limitata ha ostacolato la ricerca 1 , 2 . Molti studi dermatologici sono limitati dall&#39;inadeguata offerta, qualità variabile e costi associati all&#39;uso del tessuto umano. Il costo della creazione di una grande banca bio-dedicata è stato stimato di circa due milioni di dollari3 e questi costi collocano l&#39;uso del tessuto umano fuori dalla portata di molti ricercatori. Inoltre, il processo di generazione e conservazione di campioni di ricerca pone il rischio di influenzare le operazioni cliniche e di ritardare la cura del paziente se non eseguita con attenzione. È stato stabilito un bioreposito a basso costo basato su una clinica che si concentra sui campioni di cancro della pelle seguendo le migliori pratiche raccomandate e la convalida dei campioni 4 , 5 , 6 . <pQuesto protocollo è stato sviluppato in una clinica di dermatologia che svolge un grande volume di interventi chirurgici microscopici Mohs per rimuovere il carcinoma delle cellule squamose (SCC), il carcinoma delle cellule basali (BCC) e i tumori della pelle del melanoma. I donatori volontari possono essere reclutati da questa popolazione paziente. È importante stabilire il biorepositivo nel sito della raccolta per bloccare rapidamente tessuto e sangue da pazienti autorizzati senza ritardare il trattamento. Raccogliere campioni dalla stessa clinica minimizza le variazioni nelle tecniche di raccolta e minimizza le variazioni della qualità dei campioni, che possono essere problematiche per le applicazioni a valle 7 , 8 . L&#39;obiettivo della tecnica di chirurgia microscopica di Mohs è assicurare che tutti i tessuti del cancro siano rimossi mantenendo il più possibile il tessuto sano possibile. La procedura prevede la progressiva rimozione di strati sottili di tessuto tumorale. Ogni livello successivo è il suoEsaminato tologicamente (dopo la criocuzione del tessuto tumorale e la colorazione H &amp; E) da un dermatologo per determinare se è stato rimosso tutto il tessuto tumorale. L&#39;esclusione e l&#39;esame di strati successivi di tessuti vengono eseguiti mentre il paziente rimane nell&#39;ufficio. Questa tecnica è considerata la migliore opzione di trattamento per SCC 9 . A questo punto, la ferita è chiusa e, per migliorare la guarigione e l&#39;aspetto estetico, il tessuto normale adiacente (ANT) viene spesso eliminato. Così, questa procedura chirurgica per rimuovere un tumore è ideale per raccogliere tessuto istologicamente caratterizzato per studi futuri. La procedura di aggiudicazione dell&#39;appalto per ottenere il tessuto tumorale, i tessuti normali adiacenti, la saliva e i campioni di sangue è stata progettata per avere un impatto minimo sui compiti normali del personale ( Figura 1 ). Gli assistenti medici eseguono il sangue durante la preparazione del paziente per la procedura. Dopo la conclusione della procedura Mohs, la M<html> Lo histotechnologo di OHS prepara ulteriori diapositive istologiche del campione e trasferisce il tessuto al bioreposito. I costi associati alla creazione del bioreposito includono l&#39;acquisto di congelatori di crioconservazione, la creazione di un modesto spazio di laboratorio clinico e lo sviluppo di un programma di monitoraggio delle scorte. <img alt="Figura 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> Figura 1: sequenza di raccolta dei campioni e personale responsabile. All&#39;atto del check-in del paziente e del raggiungimento del consenso del paziente, l&#39;assistente medico raccoglie un tampone bucale e svolge un prelievo di sangue. Il dermatologo e l&#39;assistente medico poi accise il tumore e chiude la ferita, durante il quale vengono raccolti rispettivamente gli esemplari SCC e ANT. Un tecnico specializzato di laboratorio elabora il sangue e le sezioni dei campioni SCC e ANT per la coltura, la conservazione e l&#39;ingresso nel bioreposito.Ftp_upload / 55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Fare clic qui per visualizzare una versione più grande di questa figura. La diversità dei campioni raccolti consente una varietà di approcci sperimentali ( Figura 2 ). I campioni raccolti dal paziente sono tamponi bucali (saliva può anche essere raccolta se necessario), sangue intero e tessuto esclasti. I tamponi bucali e un campione di tutto il sangue vengono salvati, senza elaborazione, per la genotipizzazione e il matching dei tessuti. Il sangue intero è separato in cellule del sangue bianco (WBC) e frazioni plasmatiche per analisi future. Dopo la trasformazione di Mohs, il tumore congelato viene posto direttamente in azoto liquido e trasferito a un congelatore -80 ° C. I campioni tumorali freschi, vitali e ANT vengono coltivati ​​utilizzando modifiche delle tecniche precedenti 10 , 11 e poi criopreservate. Durante la raccolta, il numero di ciascun tipo di esempio viene registrato su un foglio di calcolo prima di entrare<html> Il programma di monitoraggio dell&#39;inventario per facilitare l&#39;elaborazione accurata ( tabella 1 ). <img alt="figura 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> Figura 2: Struttura della raccolta e dell&#39;elaborazione dei campioni biorepositivi basati su clinica. Un tampone e un campione di sangue buccale vengono raccolti dal paziente e memorizzati per il genotipizzazione a valle e la corrispondenza dei tessuti. Il sangue intero viene ulteriormente elaborato per l&#39;isolamento delle cellule del sangue bianco (WBC) e l&#39;analisi CTC, nonché per la raccolta del plasma e l&#39;analisi biopsia liquida. Il tessuto prelevato durante la procedura Mohs è trattato istologicamente per scopi diagnostici, dopo di che le diapositive istologiche possono essere utilizzate sperimentalmente per ulteriori analisi immunohistochemiche. A condizione che il campione di tessuto ecceduto sia abbastanza grande, una porzione di tessuto fresco viene rimossa e tagliata per isolamento proteico e RNA e per la creazione di linee cellulari colte.55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Fare clic qui per visualizzare una versione più grande di questa figura. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Data di raccolta: </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> Paziente 1 </td><td> Paziente 2 </td><td> Paziente 3 </td><td> Paziente 4 </td><td> Paziente 5 </td></tr><tr><td> Iniziali e data di nascita </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Colore degli occhi </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tipo di esempio </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Posizione campione </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Saliva </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> WBuco sangue </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Plasma </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumore Viable </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tessuto Normale Resistente </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumore Mohs Liquido Azoto </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tessuto normale azoto liquido </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Slides </td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> Tabella 1: Lista di controllo per registrare le collezioni di campioni. I dati tracciati e registrati con ciascun campione raccolto comprendono le iniziali del paziente, la data di nascita e il colore degli occhi (per la tipizzazione della pelle), nonché i locatiSulla rimozione del campione. Il numero di campioni salivari, i volumi di raccolta del sangue e il numero di esemplari di tessuti vitali e conservati sono registrati anche come riferimenti per le allocazioni agli usi successivi. <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">Clicca qui per scaricare questo file.</a> Per convalidare le procedure di raccolta dei campioni, ciascun tipo di esempio è stato testato nelle applicazioni a valle. Utilizzando modifiche delle tecniche precedenti 12 , il tumore e ANT sono stati utilizzati con successo nell&#39;isolamento di proteine ​​e RNA e possono essere utilizzati potenzialmente per l&#39;isolamento del DNA. Le esplosioni vitali stabilite dalle sezioni del tessuto sono state valutate mediante microscopia, mentre le diapositive istologiche memorizzate sono state utilizzate per immunohistochemistry e immunofluorescenza. Seguendo il protocollo qui descritto, è possibile estendere questo modello ad altre cliniche di dermatologia, altri tipi di tumore (come il melanoma), E altre specialità e pratiche chirurgiche per fornire campioni di tessuti umani per una ricerca multidimensionale nei tumori umani. Le modifiche leggere di questo protocollo sono probabilmente necessarie per altre pratiche, ma in linea di principio questo protocollo è applicabile a qualsiasi pratica chirurgica che scarta frequentemente i campioni dei pazienti raccolti durante il trattamento del paziente.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K3EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>02-685-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Double Edge Blades</td>
			<td>ThermoFisher Scientific</td>
			<td>50-949-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Medium Point General Purpose Forceps</td>
			<td>ThermoFisher Scientific</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza Walkersville KGM Keratinocyte Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9791321</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Tissue TEK OCT Compound</td>
			<td>ThermoFisher Scientific</td>
			<td>50-363-579</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica CM1950 Cryostat</td>
			<td>Leica Biosystems</td>
			<td>14047743909</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>ThermoFisher Scientific</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Rapid-Chrome H&#38;E Frozen Section Staining Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>99-900-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Long-Term Storage Cryogenic Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>03-337-7X</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Boca Scientific BuccalT-Swab&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9679349</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Signaling Technology 10x RIPA Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>50-195-822</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>PI78443</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>05-401-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet Morter Cordless Homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>12-141-3</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Free Pellet Pestle</td>
			<td>ThermoFisher Scientific</td>
			<td>121-141-364</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma SteriCycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>13-998-089</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>SH30071.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Advanced DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>12-634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco 1M Hepes</td>
			<td>ThermoFisher Scientific</td>
			<td>15-630-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell Culture 35 mm with Vent</td>
			<td>ThermoFisher Scientific</td>
			<td>1256591</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CFH monoclonal antibody clone OX-24</td>
			<td>Abnova</td>
			<td>MAB12583</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p40 (p63 delta) antibody</td>
			<td>Abnova</td>
			<td>ABX-144A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti MUC-1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-7313</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Snail + Slug (phospho S246)</td>
			<td>Abcam</td>
			<td>Ab63568</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Phallodin</td>
			<td>Molecular Probes&#160;</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Molecular Probes&#160;</td>
			<td>P36941</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td>
			<td>Zeiss</td>
			<td>4300009901</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX51 phase contrast with DP72 camera</td>
			<td>Olympus</td>
			<td>IX511F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinic-based biorepository

L&#39;incidenza del cancro della pelle ( es., Carcinoma delle cellule squamose , carcinoma delle cellule basali e melanoma) è in aumento negli ultimi anni. Si prevede che ci sarà una domanda parallela per campioni tumorali cutanei per studi di ricerca biomedica. La disponibilità dei tessuti, tuttavia, è limitata a causa del costo della creazione di un bioreposito e della mancanza di protocolli disponibili per ottenere campioni clinici che non interferiscono con le operazioni cliniche. È stato stabilito un protocollo per raccogliere e elaborare campioni tumorali cutanei e associati di sangue e saliva che hanno un impatto minimo sulle procedure cliniche di routine alla data di una chirurgia Mohs. I campioni tumorali vengono raccolti e trattati da pazienti sottoposti al primo strato di chirurgia Mohs per tumori maligni cutanei provenienti da biopsia da parte del histotechnologista Mohs. Il tessuto normale adiacente viene raccolto all&#39;atto della chiusura chirurgica. Ulteriori campioni che possono essere raccolti sono sangue intero e tampone buccale. Utilizzando campioni di tessuti normalmente scartati, è stato generato un bioreposito che offre diversi vantaggi fondamentali basandosi sulla clinica rispetto all&#39;ambiente di laboratorio. Questi includono una vasta gamma di campioni raccolti; L&#39;accesso ai dischi identificati, inclusi i rapporti patologici; E, per il tipico donatore, l&#39;accesso a campioni aggiuntivi durante le visite di follow-up.

Tumore e ANT sono stati utilizzati con successo in isolamento proteico e testati da blotting occidentale. Come mostrato nella figura 3 , i marker di carcinoma cutanee squamose cutanee (Muc1 14 , FHL-1 15 e p40 16,17 ) possono essere rilevati nei campioni dei pazienti raccolti e conservati nel nostro bioreposito in base alla clinica. <img al...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

I tumori cutanei vengono spesso scartati dopo la chirurgia microscopica di Mohs. Qui viene descritto un protocollo che consente al personale di supporto clinico di elaborare e memorizzare in modo efficace campioni tumorali cutanei ( ad esempio, carcinoma delle cellule squamose, carcinoma delle cellule basali e melanoma) per applicazioni di laboratorio a valle senza interferire con le operazioni cliniche.

Istituzione di un Bioreposito di base clinico

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

The overall goal of this protocol is to enable clinical support staff in a dermatology practice to effectively process and store cutaneous tumor samples for downstream laboratory applications, without interfering with routine clinical operations. This protocol was designed to enable medical assistants and Mohs histotechnicians to collect and store cutaneous tumor samples for future biomedical research projects, as part of their daily clinical operations. The main advantage of this protocol is that excised tissues that are normally discarded and destroyed are instead collected and preserved for research, using procedures, which minimally impact routine clinical operations.The variety of samples collected enables correlations between protein and RNA biomarkers in the tissue and plasma samples with patient outcome data. In principle, this protocol, once slightly modified is applicable to any surgical practice, that routinely discards patient samples, gathered in the course of patient treatment. Demonstrating the procedure will be Stefani Fawks, a Mohs histotechnician, and Dr.Mitchell Manway, a research fellow in our practice.To begin, use the tissue received from the Mohs surgeon and with a scalpel, excise a 10 to 50 milligram sample from the center of the apical side of the tissue. With tweezers, transfer the viable tissue to a labeled 1.5 milliliter tube, prefilled with culture medium. Make sure the tissue is entirely immersed in the medium.Place the sample in a refrigerator and store it at four degrees Celsius, for no longer than 24 hours before further processing. Harvest at least a 10 cubic millimeter tissue fragment from the apical side of the excised tissue, when the tumor sample is large enough, and such removal will not interfere with routine Mohs processing. Next, place the remaining unprocessed tumor sample on a cryostat tissue holder.Embed the tissue in an optimal cutting temperature compound, and freeze it within the holder to prepare histological slides. Using a cryostat, cut the tissue into seven micron thick sections, and layer up to three sections over a slide. After sectioning, fix the tissue by immersing the slides in acetone for one to two seconds.Then, allow the slides to air dry. Keeping the remaining sample in a cryostat chamber, pry the tissue from the cryo embedding medium, and transfer it to a labeled cryogenic tube. Immediately transfer the labeled vial into liquid nitrogen.Transfer of the tissue to a cryo vial after Mohs processing must be performed in the negative 20 degrees Celsius cryostat chamber. If performed properly and the tissue is kept frozen, this step will enable the successful extraction of RNA from post-Mohs tissue samples. Next, obtain adjacent normal tissue from the Mohs surgeon.Excise a sample from the apical side of the adjacent normal tissue. Then, using tweezers, transfer the excised sample to a labeled 1.5 milliliter tube filled with culture medium. Immerse the tissue in the same culture medium.Place it into an appropriate sample bag, and store at four degrees Celsius, until subsequent processing. Collect approximately 10 milliliters of venous blood into EDTA-coated collection tubes. Combine the blood samples in a sterile 15 milliliter centrifuge tube.Then, transfer approximately one milliliter of the blood sample into a 1.5 milliliter cryogenic tube, avoiding bubbles. Store this whole blood sample at minus 80 degrees Celsius, until subsequent genotyping and tissue matching. Next, centrifuge the remaining blood sample at 1000 times G for 30 minutes to obtain blood plasma.After spinning, collect and divide the plasma until one milliliter aliquots in 1.5 milliliter cryogenic tubes. And store the samples at minus 80 degrees Celsius for use during future liquid biopsy analyses. Enter the data from the checklist of patient samples, along with patient information and diagnosis, subtracted from the patient chart to the biorepository database.Finally, record the localization of the de-identified samples in the biorepository database, and move the samples to the research lab. Immediately after, transfer the tissue samples into 1.5 milliliter tubes, prefilled with appropriate extraction solutions, making sure the tissue is completely immersed. Store the samples at minus 80 degrees Celsius until further processing.Presented here are the images of representative Western blots of known squamous cell carcinoma markers, mucin one, FHL-one, and p40, obtained for adjacent normal tissue and a tumor sample. The quality of the RNA isolated from Mohs samples was first assessed using gel electrophoresis, followed by RNA integrity analysis. RNA integrity number values achieved for normal and tumor samples indicate that high quality and intact RNA was obtained.Cytometry profiles of the bands were plotted as fluorescence units versus nucleotide number for representative RNA samples from tumor and normal patient tissue. A squamous cell carcinoma tissue explant was imaged using phase contrast microscopy and shown to be a mixed keratinocyte and fibroblast culture, that might be subcultured or used for future cell line development. Finally, in the squamous cell carcinoma tissue sections, positive staining for the epithelial to mesenchymal transition marker was shown.Once mastered, this protocol will extend the standard work flow of the clinic staff to less than 30 minutes, if it is performed properly. The majority of the steps are performed by the existing clinical staff after patient care is completed. While attempting this protocol, it is important to maintain accurate record keeping, linking the samples with the patient's history and diagnosis.As this information is often needed for downstream applications. This technique will help pave the way for researchers in the field of dermatology for the development of liquid biopsies and biomarkers in squamous cell carcinomas and melanoma. After watching this video, you should have a good understanding of how to implement a clinic-based biorepository for cutaneous cancer samples, generated as part of daily practice.

establishment-of-a-clinic-based-biorepository

Research

JoVE Journal

Medicine

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Mini-Invasiva Istituzione di murini ortotopico vescica Xenotrapianti

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Ricerca

Medicina

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Istituzione di umano epiteliali Enteroids e Colonoids dal tessuto intero e biopsia

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Istituzione e caratterizzazione di UTI e CAUTI in un modello murino

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Istituzione di un segmentale femorale critico-size difetto del modello in topi stabilizzati da osteosintesi Piastra

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Istituzione di un modello di occhio secco severo utilizzando la dacrigofectomia completa nei conigli

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Istituzione e valutazione di un modello di malattia da trapianto di vena suina

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

Istituzione di un modello di ratto semplice ed efficace per l'imaging intraoperatorio delle ghiandole paratiroidi

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

Chirurgia della fistola arterovenosa modificata per stabilire il modello murino di sovraccarico del volume del ventricolo destro

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

Autore in primo piano: Istituzione e conferma di un modello murino di sovraccarico del volume ventricolare destro postnatale  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.