Supportato in parte dal NIH sovvenzione (R43/44CA122444) e DOD BRCP concessione (W81XWH-06-1-0682) per YL.

1. Campione, Attrezzatura e Preparazione dei reagenti <ol><li> Tagliare campione di tessuto in blocchi di 3-4 mm di spessore, fissare in fresco 10% formalina neutra tamponata per 16-32 ore a temperatura ambiente (25 ° C) e incorporare in paraffina. Tagliare FFPE in sezioni di 5 ± 1 micron di spessore da blocchi FFPE, montare sezioni su scivoli e aria secca. Nota: Le diapositive possono essere conservati a temperatura ambiente sotto essiccazione per un massimo di 3 mesi. I vetrini di tessuto montati dovrebbero essere cotte in un secco sopra a 60 ° C per 1 ora prima del test RNAscope. </li><li> Portare ibridazione forno a 40 ° C. Mettere una carta di umidificazione nel Umidità di controllo Cassetto. Aggiungere 50 ml di dH 2 O del Libro di umidificazione per bagnare completamente. Inserire il vassoio coperto nel forno per preriscaldare per almeno 30 minuti prima dell&#39;uso. </li><li> Rendere 700 ml 1x Pretrattare 2 Soluzione (10 nl, pH 6 tampone citrato) diluendo 10x Soluzione di pretrattamento in dH 2 O. Riscaldare il 1x Pretrattare 2 Soluzione a bollire e maintain la temperatura tra 100-104 ° C evitando sovra-bollente. </li><li> Fai 3 L 1x Wash Buffer (0.1x SSC) diluendo preriscaldata 50x Wash Buffer in dH 2 O. </li><li> Preparare 2 x 200 ml di xilene e 2 x 200 ml di 100% EtOH per deparaffinizzazione sotto una cappa aspirante. </li><li> Preparare il 50% soluzione colorante ematossilina e il reagente brunitura (ammoniaca 0,01%) per il post-colorazione sotto una cappa aspirante. </li><li> Preparare 200 ml di xilene, 200 ml di 70%, e 2 x 200 ml di EtOH 100% per disidratazione sotto una cappa aspirante. </li><li> Sonde Preriscaldare target a 40 ° C per 10 min e portare Detection RTU Kit di reagenti a temperatura ambiente, tra cui RTU Amp1, Amp2, Amp3, AMP4, Amp 5-Brown, e AMP6-Brown, tranne cromogeno DAB Brown-A e Brown-B . </li></ol> 2. RNAscope Assay Deparaffinizzazione e disidratazione Dopo la cottura, Deparaffinare sezioni di tessuto in xilene per 2 x 5 minuti con agitazione, e disidratarein 100% EtOH per 2 x 3 min sotto agitazione. Asciugare all&#39;aria per 5 min e disegnare una barriera idrofoba intorno alla sezione di tessuto con un idrofobo Barriera Pen. Pretrattamenti <ol><li> Incubare sezioni di tessuto con Pretrattare 1 per 10 min a RT per tempra di attività della perossidasi endogena, sciacquare in dH 2 O. </li><li> Incubare sezioni di tessuto con Pretrattare 2 mantenuti ad una temperatura di ebollizione per 15 minuti per il recupero di RNA, lavare due volte in dH 2 O. </li><li> Incubare sezioni di tessuto con Pretrattare 3 per 30 minuti a 40 ° C per la digestione delle proteine ​​in HybEZ forno, lavare due volte in dH 2 O. </li></ol> Obiettivo della sonda di ibridazione Sonde target HPV-HR 7 piscina includono: HPV16, 18, 31, 33, 35, 52, e 58. Aggiungi sonde HPV, ubiquitina C (UBC) e batteriche sonde gene dapB separatamente su tre sezioni di tessuto adiacenti. Ibridare a 40 ° C in forno per 2hr, quindi risciacquare in 1x tampone di lavaggio per 2 x 2 min a RT. Amplificazione del segnale <ol><li> Incubare sezioni di tessuto con Amp1 (preamplificatore) per 30 min a 40 ° C, Amp2 (sfondo riduttore) per 15 min a 40 ° C, Amp3 (amplificatore) per 30 min a 40 ° C, e AMP4 (sonda etichetta) per 15 min a 40 ° C in forno HybEZ. Dopo ogni fase di ibridazione, sciacquare in 1x tampone di lavaggio per 2 x 2 min a RT. </li><li> Incubare sezioni di tessuto con AMP5 per 30 min e AMP6 per 15 minuti a RT. Dopo ogni fase di incubazione, sciacquare in 1x tampone di lavaggio per 2 x 2 min a RT. </li></ol> Rilevamento segnale Incubare sezioni di tessuto con il 1:1 DAB Miscela miscelando un volume uguale di Brown-A e Brown-B per 10 minuti a RT, lavare due volte in dH 2 O. Di contrasto <pclass = &quot;jove_content&quot;&gt; sezioni di tessuto macchia con soluzione di ematossilina al 50% per 2 min a RT, sciacquare con dH 2 O fino a quando le diapositive sono chiare mentre il tessuto resta viola. Immergere i vetrini in 0,01% di ammoniaca in dH 2 O per 5x e seguita con 5 tuffi in dH 2 O. Cursore di montaggio Disidratare sezioni di tessuto a 70%, 100% e 100% EtOH per 2 min ciascuna, xilene per 5 min, montare con un mezzo di montaggio a base di xilene.

<ol>
	<li>Parkin, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+health+burden+of+infection-associated+cancers+in+the+year+2002.">The global health burden of infection-associated cancers in the year 2002.</a> Int. J. Cancer. 118 (12), 3030-3044 (2006).</li><li>Chaturvedi, A. K., 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=Human+papillomavirus+and+rising+oropharyngeal+cancer+incidence+in+the+United+States.">Human papillomavirus and rising oropharyngeal cancer incidence in the United States.</a> J. Clin. Oncol. 29 (32), 4294-4301 (2011).</li><li>Ang, K. K., 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=Human+papillomavirus+and+survival+of+patients+with+oropharyngeal+cancer.">Human papillomavirus and survival of patients with oropharyngeal cancer.</a> N. Engl. J. Med. 363 (1), 24-35 (2010).</li><li>Smeets, S. 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=A+novel+algorithm+for+reliable+detection+of+human+papillomavirus+in+paraffin+embedded+head+and+neck+cancer+specimen.">A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen.</a> Int. J. Cancer. 121 (11), 2465-2472 (2007).</li><li>Wang, F., 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J. Mol. Diagn. 14 (1), 22-29 (2012).</li><li>Liu, X., 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=Specific+regulation+of+NRG1+isoform+expression+by+neuronal+activity.">Specific regulation of NRG1 isoform expression by neuronal activity.</a> J. Neurosci. 31 (23), 8491-8501 (2011).</li><li>Yan, K. 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=The+intestinal+stem+cell+markers+Bmi1+and+Lgr5+identify+two+functionally+distinct+populations.">The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (2), 466-471 (2012).</li><li>Payne, R. E., 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=Viable+circulating+tumour+cell+detection+using+multiplex+RNA+in+situ+hybridisation+predicts+progression-free+survival+in+metastatic+breast+cancer+patients.">Viable circulating tumour cell detection using multiplex RNA in situ hybridisation predicts progression-free survival in metastatic breast cancer patients.</a> Br. J. Cancer. 106 (11), 1790-1797 (2012).</li><li>Bordeaux, J. 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=Quantitative+in+situ+measurement+of+estrogen+receptor+mRNA+predicts+response+to+tamoxifen.">Quantitative in situ measurement of estrogen receptor mRNA predicts response to tamoxifen.</a> PLoS One. 7 (5), (2012).</li><li>Wang, Z., 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=Automated+quantitative+RNA+in+situ+hybridization+for+resolution+of+equivocal+and+heterogeneous+ERBB2+(HER2)+status+in+invasive+breast+carcinoma.">Automated quantitative RNA in situ hybridization for resolution of equivocal and heterogeneous ERBB2 (HER2) status in invasive breast carcinoma.</a> J. Mol. Diagn. 15 (2), 210-219 (2013).</li><li>Tubbs, R. R., 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=Ultrasensitive+RNA+in+situ+Hybridization+for+Detection+of+Restricted+Clonal+Expression+of+Low+Abundance+Immunoglobulin+Light+Chain+mRNA+in+B-Cell+Lymphoproliferative+Disorders.">Ultrasensitive RNA in situ Hybridization for Detection of Restricted Clonal Expression of Low Abundance Immunoglobulin Light Chain mRNA in B-Cell Lymphoproliferative Disorders.</a> Am. J. Surg. Pathol. In press, (2013).</li><li>Ukpo, O. C., Flanagan, J. J., Ma, X. J., Luo, Y., Thorstad, W. L., Lewis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-risk+human+papillomavirus+E6/E7+mRNA+detection+by+a+novel+in+situ+hybridization+assay+strongly+correlates+with+p16+expression+and+patient+outcomes+in+oropharyngeal+squamous+cell+carcinoma.">High-risk human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma.</a> Am. J. Surg. Pathol. 35 (9), 1343-1350 (2011).</li><li>Lewis, J. 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=Transcriptionally-active+high-risk+human+papillomavirus+is+rare+in+oral+cavity+and+laryngeal/hypopharyngeal+squamous+cell+carcinomas--a+tissue+microarray+study+utilizing+E6/E7+mRNA+in+situ+hybridization.">Transcriptionally-active high-risk human papillomavirus is rare in oral cavity and laryngeal/hypopharyngeal squamous cell carcinomas--a tissue microarray study utilizing E6/E7 mRNA in situ hybridization.</a> Histopathology. 60 (6), 982-991 (2012).</li><li>Lewis, J. 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=Partial+p16+staining+in+oropharyngeal+squamous+cell+carcinoma:+extent+and+pattern+correlate+with+human+papillomavirus+RNA+status.">Partial p16 staining in oropharyngeal squamous cell carcinoma: extent and pattern correlate with human papillomavirus RNA status.</a> Mod. Pathol. 25 (9), 1212-1220 (2012).</li><li>Bishop, J. 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=Detection+of+transcriptionally+active+high-risk+HPV+in+patients+with+head+and+neck+squamous+cell+carcinoma+as+visualized+by+a+novel+E6/E7+mRNA+in+situ+hybridization+method.">Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method.</a> Am. J. Surg. Pathol. 36 (12), 1874-1882 (2012).</li><li>Schache AG, ., 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=Validation+of+a+novel+diagnostic+standard+in+HPV-positive+oropharyngeal+squamous+cell+carcinoma.">Validation of a novel diagnostic standard in HPV-positive oropharyngeal squamous cell carcinoma.</a> Br. J. Cancer. 108 (6), 1332-1339 (2013).</li><li>Mehrad, 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=Papillary+Squamous+Cell+Carcinoma+of+the+Head+and+Neck:+Clinicopathologic+and+Molecular+Features+With+Special+Reference+to+Human+Papillomavirus.">Papillary Squamous Cell Carcinoma of the Head and Neck: Clinicopathologic and Molecular Features With Special Reference to Human Papillomavirus.</a> Am. J. Surg. Pathol. Jun. , (2013).</li><li>Jordan, R. C., 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=Validation+of+methods+for+oropharyngeal+cancer+HPV+status+determination+in+US+cooperative+group+trials.">Validation of methods for oropharyngeal cancer HPV status determination in US cooperative group trials.</a> Am. J. Surg. Pathol. 36, 945-954 (2012).</li><li>Hammond, M. E., 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=American+Society+of+Clinical+Oncology/College+of+American+Pathologists+guideline+recommendations+for+immunohistochemical+testing+of+estrogen+and+progesterone+receptors+in+breast+cancer+(unabridged+version).">American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version).</a> Arch. Pathol. Lab. Med. 134 (7), 48-72 (2010).</li></ol>

Tutti gli autori sono impiegati da e proprie azione in Advanced Diagnostics cella, Inc.

Il test HPV RNAscope permesso la visualizzazione diretta di E6/E7 mRNA in situ in testa HPV-associata collo e carcinoma a cellule squamose. Il saggio RNAscope è pienamente compatibile con il tessuto tumorale fisso di routine e conserva la morfologia del tessuto per correlazioni istopatologiche (Figura 2). Uno dei principali vantaggi del test RNAscope rispetto ai metodi convenzionali CISH è che amplifica specificamente i segnali di ibridazione (Figure 2B e 2E), senza amplificare il rumore di fondo (Figure 2C e 2F). In pratica, la procedura di analisi RNAscope HPV può essere completato entro 8 ore o comodamente divisa in due giorni. Il saggio RNAscope HPV è stato utilizzato per determinare lo stato di HPV in testa e del collo carcinoma a cellule squamose 12-16, dimostrando il 97% di sensibilità e specificità del 93% utilizzando qRT-PCR come metodo di riferimento 16. Chr convenzionaleISH omogenic per HR-HPV DNA è altamente specifica, ma ha una sensibilità del 12 ~ 80%. Immunoistochimica (IHC) colorazione per il cellulare surrogato marcatore p16 dimostra sensibilità eccellente, ma può generare risultati falsi positivi 15,18, soprattutto in nonoropharyngeal tumori testa e collo 15. L&#39;attuale metodo &quot;gold standard&quot; di qRT-PCR per l&#39;HPV E6/E7 mRNA rilevamento richiede tessuto fresco congelato per ottenere risultati ottimali ed è tecnicamente complessa, che ne limita l&#39;utilizzo solo al laboratorio di ricerca. Inoltre, richiede l&#39;estrazione di RNA che rende impossibile correlare espressione HR-HPV E6/E7 mRNA con istopatologia. Ci sono diversi fattori critici per il successo del saggio RNAscope. In primo luogo, per i migliori risultati, i tessuti dovrebbero essere fissati in fresco 10% formalina neutra tamponata a temperatura ambiente per 16-32 ore secondo le linee guida ASCO / CAP 19. In secondo luogo, il forno HybEZ è altamente raccomandato in quanto consentonos controllo ottimale della temperatura e dell&#39;umidità per sonda di ibridazione e gradini amplificazione del segnale. In terzo luogo, è importante rimuovere buffer residuo in eccesso prima di ogni passo, ma ancora mantenere la sezione di tessuto da asciugare durante tali operazioni. La procedura RNAscope manuale qui descritto è stato completamente automatizzato su un vetrino sistema di auto-colorazione commerciali 10. Ciò dovrebbe facilitare notevolmente la standardizzazione delle condizioni di saggio e di risparmiare prezioso lavoro manuale nei laboratori di patologia clinica. Inoltre, il software di analisi di immagine dedicato è stato sviluppato 10 per identificare automaticamente le cellule e segnali di colorazione su un vetrino digitalizzato, che dovrebbe contribuire ad eliminare soggettività e migliorare la riproducibilità nel punteggio. In sintesi, il saggio RNAscope HPV rileva la presenza di trascritti HR-HPV E6/E7 mRNA in situ nei tessuti FFPE. Ha un flusso di lavoro che è familiare a patologia clinica laboratori per consentire la diretta visualizzazione di questi in sezioni di tessuto. Esso offre una piattaforma ideale per l&#39;esame (FFPE) campioni di tessuto, e può essere facilmente adottata dai laboratori diagnostici di patologia.

Conti di papillomavirus umano ad alto rischio (HR-HPV) per circa il 5% di tutti i tumori in tutto il mondo 1. L&#39;incidenza del cancro orofaringeo HPV-associata è aumentata negli ultimi decenni, soprattutto tra gli uomini. Orofaringeo a cellule squamose HPV-positive (OPSCC) probabilmente costituirà la maggioranza di tutti i tumori della testa e del collo negli Stati Uniti nei prossimi 20 anni 2. Orofaringei carcinomi a cellule squamose causati da HPV sono associati con la sopravvivenza favorevole, e lo stato del tumore HPV è un fattore prognostico forte e indipendente per la sopravvivenza 3. Prove per l&#39;attivazione trascrizionale di oncogeni virali E6 ed E7 è considerato il gold standard per la presenza di HPV clinicamente rilevante 4. Tuttavia, la scoperta di E6/E7 mRNA da tessuto tumorale fresco mediante tecnica RT-PCR non è pratico nella pratica clinica ed è stato limitato ai laboratori di ricerca. Recentemente, abbiamo HAVe ha sviluppato una nuova tecnologia chiamata RNA ISH RNAscope, che consente il rilevamento multiplex in celle singole, con il singolo di RNA sensibilità molecola nei campioni in formalina paraffina fissati incorporati tessuti (FFPE) 5-10. Abbiamo fornito tre linee di evidenza per singola molecola di rilevamento 5. In primo luogo, la progettazione e segnale del sistema di amplificazione sonda RNAscope permesso di rilevamento singola copia genomiche HER2 bersagli di DNA in HeLa e SK-BR-3 linee cellulari. In secondo luogo, se confrontato con HER2 segnali di DNA genomico, la distribuzione delle intensità di fluorescenza di punti di segnale HER2 mRNA in cellule HeLa era coerente con una molecola per punto. In terzo luogo, il numero di punti di segnale HER2 mRNA per cella corrisponde strettamente il numero di copie HER2 mRNA stimato mediante un saggio quantificazione basata soluzione, sostenendo ulteriormente rilevamento singola molecola. Inoltre, di contrasto dei nuclei con DAPI in microscopia a fluorescenza o con ematossilina in microscopia in campo chiaro permette la visualizzazione dei singoli nuclei, which a sua volta consente il rilevamento e la quantificazione degli obiettivi di RNA su base singola cellula 10. La capacità di analizzare l&#39;espressione genica in situ in campioni clinici di routine rende RNAscope una piattaforma promettente per patologia diagnostica, in particolare quelli di tessuto FFPE sezione basata saggi 10,11. Abbiamo sviluppato un saggio basato HPV-RNAscope per rilevare E6/E7 mRNA di sette genotipi ad alto rischio di HPV (HPV16, 18, 31, 33, 35, 52, e 58) utilizzando un pool di sonde genotipo-specifici. I nostri recenti studi in OPSCC hanno dimostrato che il saggio RNAscope HPV è altamente sensibile e specifico per determinare lo stato di HPV sui tessuti FFPE 12-17, e anche informa la prognosi in OPSCC 12,16. Il principio della tecnologia RNAscope è stato descritto in precedenza 5. Qui, descriviamo il protocollo completo di analisi RNAscope e dimostrare il suo utilizzo nella rilevazione di HR-HPV in FFPE sezioni di tessuto tumorale.

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<table border="0" cellpadding="0" cellspacing="0" width="580">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="39">
			<td height="60" rowspan="2" style="height:60px;width:145px;">SuperFrost Plus slides</td>
			<td rowspan="2" style="width:145px;">Fisher Scientific</td>
			<td rowspan="2" style="width:145px;">12-550-15</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:145px;">HybEZ Oven, Tray, and Rack</td>
			<td style="width:145px;">Advanced Cell Diagnostics</td>
			<td style="width:145px;">310011, 310012, 310014</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:145px;">RNAscope 2.0 FFPE Reagent Kit - Brown</td>
			<td style="width:145px;">Advanced Cell Diagnostics</td>
			<td style="width:145px;">310035</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:145px;">RNAscope HPV-HR7 Probe for HPV 16,18,31,33,35,52 and 58, E6/E7 mRNA</td>
			<td style="width:145px;">Advanced Cell Diagnostics</td>
			<td style="width:145px;">312351</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:145px;">ImmEdge Hydrophobic Barrier Pen</td>
			<td style="width:145px;">Vector Laboratory</td>
			<td style="width:145px;">H-4000</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:145px;">100% EtOH</td>
			<td rowspan="2" style="width:145px;">American Master Tech Scientific</td>
			<td rowspan="2" style="width:145px;">ALREAGAL</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:145px;">Xylene&#160;</td>
			<td rowspan="2" style="width:145px;">Fisher Scientific</td>
			<td rowspan="2" style="width:145px;">X3P-1GAL</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:145px;">Gill&#8217;s Hematoxylin I</td>
			<td rowspan="2" style="width:145px;">American Master Tech Scientific</td>
			<td rowspan="2" style="width:145px;">HXGHE1LT</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:145px;">Ammonia hydroxide</td>
			<td rowspan="2" style="width:145px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:145px;">320145-500mL</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:145px;">Cover Glass 24x50 mm</td>
			<td rowspan="2" style="width:145px;">Fisher Scientific</td>
			<td rowspan="2" style="width:145px;">12-545-F</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:145px;">Hot Plate</td>
			<td rowspan="2" style="width:145px;">Fisher Scientific</td>
			<td rowspan="2" style="width:145px;">11-300-49SHP</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="99">
			<td height="120" rowspan="2" style="height:120px;width:145px;">Drying Oven</td>
			<td rowspan="2" style="width:145px;"></td>
			<td rowspan="2" style="width:145px;"></td>
			<td rowspan="2" style="width:145px;">Capable of holding temperature at 60 &#177; 1&#176;C</td>
		</tr>
		<tr height="21">
		</tr>
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			<td height="120" rowspan="2" style="height:120px;width:145px;">Water Bath</td>
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			<td rowspan="2" style="width:145px;"></td>
			<td rowspan="2" style="width:145px;">Capable of holding temperature at 40 &#177; 1&#176;C</td>
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rnascope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma

Il &#39;gold standard&#39; per la rilevazione di HPV oncogeni è la dimostrazione di trascrizionalmente attivo HPV ad alto rischio nel tessuto tumorale. Tuttavia, l&#39;individuazione di E6/E7 mRNA da parte quantitativa trascrizione inversa reazione a catena della polimerasi (qRT-PCR) richiede l&#39;estrazione di RNA che distrugge il tessuto tumorale contesto critico per la correlazione morfologica ed è stato difficile da adottare nella pratica clinica di routine. Il nostro RNA recentemente sviluppato in tecnologia situ ibridazione, RNAscope, permette la visualizzazione diretta di RNA in, inclusi in paraffina (FFPE) tessuto fissato in formalina con la sensibilità di singola molecola e la risoluzione singola cellula, che consente altamente sensibile e specifica analisi in situ di un biomarker RNA in campioni clinici di routine. Il saggio RNAscope HPV è stato progettato per rilevare la E6/E7 mRNA di sette genotipi HPV ad alto rischio (HPV16, 18, 31, 33, 35, 52, e 58) con un pool di sonde genotipo-specifici. Si è dimostrato eccellente sensibilitàe specificità contro l&#39;attuale metodo &#39;gold standard&#39; per l&#39;individuazione di E6/E7 mRNA da qRT-PCR. Stato determinato da HPV RNAscope è fortemente prognostico di outcome clinico nei pazienti con cancro orofaringeo.

Workflow test HPV RNAscope Il saggio RNAscope ha un flusso di lavoro altamente semplificato che è simile a IHC (Figura 1). Si compone di quattro fasi principali: pretrattamenti, ibridazione, amplificazioni del segnale, e rilevamento. Si avvale di una strategia progettuale sonda unico che garantisce il segnale ad alta fedeltà di amplificazione 5. Nel saggio RNAscope HPV, sette probe set HR-HPV sono raggruppati, tutti riconosciuta dallo stesso sistema di amplificazione di segnale collegato a perossidasi di rafano (HRP). Segnali di ibridazione specifiche vengono rilevati come precipitati marroni formate da HRP catalizzata reazione cromogena DAB usando come substrato, che può essere facilmente visualizzato mediante microscopia a campo chiaro standard. Colorazione Rappresentante per la rilevazione dell&#39;HPV La figura 2 mostra immagini di esempio da macchiati sezioni FFPE della testa e del collo, carcinoma a cellule squamose. Nel caso HPV-positive (Fig.ure 2A), le sonde HR-HPV rilevate forti segnali puntiformi specificamente nelle cellule tumorali. La sonda UBC rilevato numerosi segnali citoplasmatici puntiformi in entrambe le cellule tumorali e cellule stromali (Figura 2B). La sonda gene batterico dapB dimostrato un fondo pulito (Figura 2C). Nel caso HPV-negative, sia la sonda HR-HPV e la sonda dapB rilevati segnali (figure 2D e 2F), mentre segnali forti sono stati rilevati utilizzando la sonda UBC (Figura 2E). In questo saggio, UBC serve come controllo positivo per valutare la qualità del tessuto RNA e dapB come controllo negativo per segnali di fondo. Il punteggio per la determinazione dello status HPV comporta l&#39;esame tutte e tre le diapositive per ciascun caso. Il livello di colorazione sul vetrino di controllo negativo (dapB) cursore serve come cutoff: positività HPV è definita dalla presenza di punctate citoplasmatica e / o colorazione nucleare che era sopra il segnale sul vetrino dabpB. <pclass = &quot;jove_content&quot; fo: keep-together.within-page = &quot;always&quot;&gt; <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51426/51426fig1highres.jpg" src="/files/ftp_upload/51426/51426fig1.jpg" /> Figura 1. Diagramma di flusso del test RNAscope. Illustrazione di workflow dosaggio RNAscope. Il saggio RNAscope contiene 4 fasi principali, pretrattamenti, sonda obiettivo ibridazione, amplificazione del segnale e la rilevazione del segnale. L&#39;intera procedura di dosaggio può essere completato in 8 ore. <a href="https://www.jove.com/files/ftp_upload/51426/51426fig1highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita.</a> <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/51426/51426fig2highres.jpg" src="/files/ftp_upload/51426/51426fig2.jpg" /> Figura 2. Immagini di esempio di diapositive RNAscope-colorati. Sezioni FFPE colorate con sonde per HR-HPV, UBC (controllo positivo) e dapB (controllo negativo)da due casi HNSCC. AC. Caso HPV-positive. A, espressione HR-HPV E6/E7 mRNA in cellule tumorali. Inset, 40X di ingrandimento per mostrare i segnali puntiformi. B e C) sezioni di tessuto adiacenti che mostrano una colorazione positiva UBC (B) e negativo per dapB (C), rispettivamente. DF) HPV-negative caso. D) nessuna colorazione per l&#39;HPV E6/E7 mRNA , simile al controllo negativo dapB (F). E) UBC colorazione positiva. <a href="https://www.jove.com/files/ftp_upload/51426/51426fig2highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita.</a>

Watch this Scientific Journal Video about RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma at JoVE.com

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

La presenza di HPV ad alto rischio in testa e del collo tessuto tumorale è associata a esiti favorevoli. L&#39;RNA recentemente sviluppato nella tecnica di ibridazione in situ chiamata RNAscope permette la visualizzazione diretta di HPV E6/E7 mRNA in sezioni di tessuto FFPE.

RNAscope per<em&gt; In situ</em&gt; Rilevamento di trascrizionale attivo Papillomavirus Umano in Cervico Carcinoma a cellule squamose

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

rnascope-for-situ-detection-transcriptionally-active-human

Research

JoVE Journal

Medicine

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

27.0K Views.  Advanced Cell Diagnostics, Inc..  La presenza di HPV ad alto rischio in testa e del collo tessuto tumorale è associata a esiti favorevoli. L'RNA recentemente sviluppato nella tecnica di ibridazione in situ chiamata RNAscope permette la visualizzazione diretta di HPV E6/E7 mRNA in sezioni di tessuto FFPE. 

Video: RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Ricerca

Medicina

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Cultura primario di Human vestibolare Schwannomi

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

Il<em&gt; In Ovo</em&gt; Chick corioallantoidea membrana (CAM) Saggio come un efficiente dello xenotrapianto Modello di carcinoma epatocellulare

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

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a &#34;side population&#34; (SP). As the SP cells represent a low percentage (&#60;5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of &#946;-catenin.

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

Isolamento e caratterizzazione di un testa e del collo a cellule squamose Carcinoma sottopopolazione Avere Stem Cell Caratteristiche

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

Approccio del canale semicircolare posteriore per consegna del Gene dell'orecchio interno in Mouse neonatale

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

&lt;em&gt;In Vivo&lt;/em&gt; Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&amp;#232;re&#039;s Disease Ears and Normal Hearing Ears

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

In Vivo Analisi morfometrica dei nervi cranici umano usando la formazione immagine a risonanza magnetica in orecchie di malattia di Menière e orecchie udito normale

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Porcine As a Training Module for Head and Neck Microvascular Reconstruction

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes have been available for years in many surgical fields. We used the female (because they are easy to handle for the procedure) Yorkshire pigs for the head and neck reconstruction by harvesting the deep inferior epigastric artery perforator or the superior epigastric artery perforator flap. The anastomosis site (neck skin defect or tracheal wall defect) was prepared via the dissection of the common carotid artery and the internal jugular vein, in which 3.5&#215; loupe magnification was used for anastomosis as we use on human cases in real life. This procedure demonstrates a new training method using a reliable learning model and provides a detailed anatomy in a live scenario. We focused on the ischemia time, harvesting, vessel anastomosis, and designing the flap to fit the defect site. This model improves tissue handling and with the use of proper instruments can be repeated many times so that the surgeon is fully confident before starting the surgery on humans.

Here we present a protocol for the use of the pig superior epigastric artery perforator flap as a learning module for head and neck microvascular reconstruction.

Porcino come un modulo di formazione per ricostruzione microvascolare del collo e testa

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Il modello Chick Chorioallantoic Membrane In Vivo per valutare l'invasione perineurale nel cancro alla testa e al collo

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Learning Modern Laryngeal Surgery in a Dissection Laboratory

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is almost completely reserved to a few referral centers that deal with a large proportion of this pathology. Practice on human specimens is not always possible for ethical, economic, or availability reasons. The aim of this study is to provide a reproducible method for the organization of a laryngeal laboratory on ex vivo animal models where it is possible to approach, learn, and refine laryngeal techniques. Porcine and ovine larynges are ideal, affordable, models to simulate laryngeal surgery given their similarity to the human larynx in their anatomical layout and tissue composition. Herein, the surgical steps of transoral laser surgery, open partial horizontal laryngectomy, and total laryngectomy are reported. The merging of endoscopic and exoscopic views guarantees an inside-out perspective, which is vital for the comprehension of the complex laryngeal anatomy. The method was successfully adopted during three sessions of a dissection course &#34;Lary-Gym&#34;. Further perspectives on robotic surgical training are described.

The purpose of this paper is to illustrate how to organize a reproducible laboratory for laryngeal surgery on affordable and closely similar animal laryngeal models in order to improve anatomical and surgical knowledge and skills.

Imparare la chirurgia larigole moderna in un laboratorio di dissezione

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Preparazione di impalcature renali decellularizzate nei ratti

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...