The authors would like to gratefully thank Steffen Lippert, Khalid Aliyev, Korinna J&#246;hrens and Katharina Struecker for their help during this project.

Gli animali sono stati tenuti presso l&#39;impianto di Medicina Sperimentale (FEM, Charité, Berlino, Germania), e tutti i protocolli sperimentali sono stati esaminati e approvati dall&#39;Ufficio di Stato per gli Affari Salute e locali (LAGeSo, Berlino, Germania; Reg. No. O 0365 / 11). 1. Fegato raccolta <ol><li> Preparazione pre-chirurgica <ol><li> Utilizzare una piastra di sughero per il fissaggio. Mettere un drappo medico sul piatto. Utilizzando quattro aghi, fissare una maschera di inalazione sulla piastra di sughero per intraoperatoria inalazione narcosi. </li></ol></li><li> Predisposizione del Portale venoso cannula <ol><li> Collegare un catetere venoso periferico (G 20) ad un rubinetto a tre vie. Il catetere venoso deve essere tagliato obliquamente, con una lunghezza di circa. 1,5 centimetri. </li><li> De-air sistema utilizzando una siringa da 10 ml contenente una soluzione Ringer. E &#39;molto importante per cura de-aria, perché embolia gassosa può influenzare negativamente il decellularization perfusione. </li></ol></li><li> Predisposizionedella cannula arteriosa <ol><li> Usare una cannula farfalla e tagliare l&#39;ago ad una lunghezza di 5 mm. Montare un tubo di 5 centimetri (ID 0,58 millimetri; diametro 0,96 millimetri) per l&#39;ago 5 mm e fissarlo con colla. Inserire una provetta 2 cm di lunghezza (ID 0,28 millimetri; diametro 0,61 millimetri) nel tubo più grande e fissarlo con colla. </li><li> Infilare un altro tubo su questa costruzione da riscontro e fissarlo con colla. De-air cannula arteriosa con una siringa da 5 ml riempito con soluzione di Ringer. </li></ol></li><li> Anestesia e Analgesia <ol><li> Indurre narcosi del topo nella camera di induzione di anestesia del 3,5% isoflurano in ossigeno ad un flusso di 1,5 l / min. </li><li> Per garantire l&#39;analgesia sicuro durante il funzionamento, iniettare metamizol (100 mg / kg di peso corporeo) e basse dosi di ketamina / medetomidina (10 mg / 0,1 mg per kg di massa corporea) per via intraperitoneale. </li><li> Fornire 1,5% isoflurano in 1 L / min di ossigeno attraverso la maschera di inalazione per il successivo mantenimento dell&#39;anestesia chirurgica. </li></ol></li><li> Accordi preoperatori &lt;ol&gt; <li> Radere l&#39;addome liberamente con una macchina di taglio elettrica. Inumidire il sito di incisione rasata sull&#39;addome con il 70% di etanolo per la disinfezione. </li><li> Posto l&#39;animale in posizione supina sul piatto preparato, inserire la testa nella maschera di inalazione e fissare le arti con nastro medico e perni. Rimuovere l&#39;eccesso di etanolo e pelliccia con un impacco di garza. </li></ol></li><li> Anestesia Assessment <ol><li> Valutare la profondità dell&#39;anestesia guardando la frequenza respiratoria. Quando l&#39;anestesia sembra abbastanza profondo (40-60 respiri / min), controllare che l&#39;analgesia sia sufficiente cercando di attivare il riflesso pizzico punta con pinze chirurgiche per pizzicare la pelle tra le dita di entrambi gli arti posteriori. </li></ol></li><li> Approccio addominale <ol><li> Effettuare una incisione mediana nella parete addominale caudale sopra la vescica. Tagliare da questo punto in entrambe le arcate costali laterali in modo a forma di V. Fate attenzione a non tagliare nel diaframma. Tirare la parete addominale tagliata sul petto. </li&gt; <li> Fissare il xifoideo con un morsetto Overholt, tirarlo craniale e fissarlo con un elastico. Usare tamponi di cotone umido per evacuare l&#39;intestino. Avvolgere l&#39;intestino in una benda di garza umida. Inoltre, coprire tutti i margini addominale, con un bendaggio umido. </li></ol></li><li> Fegato Preparazione <ol><li> Sezionare il legamento falciforme. Utilizzare una benda di garza bagnata per contenere i lobi del fegato mediali e sinistra craniale sotto la cupola del diaframma. Esporre il legamento epatoduodenale e il lobo epatico omentale superiore. </li><li> Utilizzare due microforceps e forbici primavera di sezionare con cura il legamento attaccato al lobo omentale superiore fino al lobo è mobile. Spostare il lobo utilizzando tamponi di cotone bagnato sotto l&#39;impacco di garza, tenendo i lobi mediali e lasciate in luogo. </li><li> Dopo la mobilitazione del lobo epatico omentale superiore, spostare lo stomaco verso sinistra. Fissare lo stomaco con un morsetto e sezionare il omento minore con microforceps e le forbici primavera. </li><li> Mobilitare la omenta bassal lobo epatico finché non è mobile e successivamente spostare il lobo indietro nella sua cavità. Utilizzare un morsetto Backhaus per mantenere il duodeno. Spostare il duodeno verso sinistra per allungare ed esporre il legamento epatoduodenale. Circoscrivere il dotto biliare comune nel legamento epatoduodenale allungato. </li><li> Sezionare il dotto biliare dal adiposo e tessuto pancreatico. Tagliare il dotto biliare 1,5 cm dal biforcazione dotto biliare. Sezionare la vena porta dal tessuto adiposo circostante. Esporre la vena gastroduodenale. </li><li> Legare la vena gastroduodenale due volte con la seta 6-0 e dividere la vena tra i 2 legature. Sezionare il tronco celiaco e l&#39;arteria epatica comune dal tessuto circostante fino a quando tutti rami arteriosi si rivelano. </li><li> Liberare l&#39;arteria gastroduodenale dal tessuto circostante. Tie l&#39;arteria gastroduodenale due volte con seta 6-0, con i legami distanti tra loro. Sezionare l&#39;arteria gastroduodenale tra i 2 legature. </li><li> Liberate splenicadal tessuto circostante. Tie splenica due volte con seta 6-0, con i legami distanti tra loro. Sezionare splenica tra i 2 legature. </li><li> Liberare l&#39;arteria gastrica sinistra dal tessuto circostante. Tie l&#39;arteria gastrica sinistra due volte con seta 6-0, con i legami distanti tra loro. Sezionare l&#39;arteria tra i 2 legature. </li><li> Circoscrivere la vena cava inferiore tra il rene destro e il lobo destro del fegato laterale. Sezionare la vena dallo spazio retroperitoneale. Esporre l&#39;aorta seguendo il tronco celiaco. </li><li> Sezionare il tessuto adiposo retroperitoneale. Iniettare 1.000 eparina IE in 1 ml di soluzione salina nella vena cava inferiore. Ritrarre la cannula e chiudere l&#39;incisione con un batuffolo di cotone. Attendere 1-2 min per l&#39;effetto sistemico dell&#39;eparina. </li><li> Circoscrivere l&#39;aorta con una pinza. Staccare l&#39;aorta e Exsanguinate ratto. Inoltre, sezionare la vena cava inferiore distale dal rene. </li></ol></li><li> Portale venosa Incannulazione (Usa la cannula preparata nella sezione 1.2) <ol><li> Effettuare una incisione pesce-bocca nella vena porta distale. Aprire l&#39;incisione con una pinza e incannulare la vena porta. Lavare il fegato con una soluzione di 20 ml Ringer fino agli decolorizes fegato e fissare la cannula con legature (seta 6-0). </li></ol></li><li> Arterioso Incannulazione (Usa la cannula preparata nella sezione 1.3) <ol><li> Sezionare l&#39;aorta sopra il tronco celiaco. Liberate il segmento dell&#39;aorta dallo spazio retroperitoneale. Tagliare il segmento dell&#39;aorta longitudinalmente per generare una patch aortica. Inserire la cannula auto-costruito in un supporto statico. </li><li> Utilizzare 2 pinze a scivolare la patch aortica sopra la cannula. Legare l&#39;arteria epatica con la seta 6-0 sopra la cannula e fissare la patch il pilastro. </li></ol></li><li> Fegato espianto <ol><li> Aprire il diaframma, fare un&#39;incisione circolare e sezionare il fegato dalle strutture circostanti. </li></ol></li><li> Immagazzinamento <ol><li> Store il fegato in una sterile serbatoio 500 ml riempito con 350 ml soluzione Ringer fino al momento dell&#39;uso. </li></ol></li></ol> 2. detergenti Solutions <ol><li> Soluzione al 10% Triton X-100 magazzino <ol><li> Riempire una bottiglia da 1.000 ml con 900 ml di acqua distillata. e aggiungere 100 ml di Triton X-100 (99,9%). Utilizzare un agitatore per sciogliere il Triton X-100. Riempire la bottiglia con acqua distillata. fino ad un volume totale di 1000 ml viene raggiunto. </li></ol></li><li> 1% Triton X-100 <ol><li> Aggiungere 100 ml della soluzione madre (10%) per un flacone 1000 ml. Aggiungere 900 ml di acqua distillata. e agitare la bottiglia per due volte. Filtrare la soluzione Triton X-100 1% attraverso un filtro sterile. </li></ol></li><li> 10% SDS Stock Solution <ol><li> Riempire una bottiglia da 1.000 ml con 900 ml di acqua distillata. e aggiungere 66.99 g SDS (99,9%, di densità 0,67). Utilizzare un agitatore per sciogliere la SDS. Riempire la bottiglia con acqua distillata. fino ad un volume totale di 1000 ml viene raggiunto. </li></ol></li><li> 1% SDS <ol><li> Aggiungere 100 ml di soluzione di riserva (10%) per un bot 1.000 mlTLE. Aggiungere 900 ml di acqua distillata. e agitare la bottiglia per due volte. Filtrare la soluzione SDS 1% attraverso un filtro sterile. </li></ol></li></ol> 3. Decellularization <ol><li> Set-up del dispositivo di perfusione <img alt="Figura 1" src="/files/ftp_upload/53029/53029fig1.jpg" /></li></ol> Figura 1:. Schema del dispositivo di perfusione selettiva per arterioso e venoso portale Perfusione di Quattro Rat Livers sotto oscillanti Condizioni Pressione (. Modificato da Struecker et al 14) <ol><li> Collegare lo scarico di un rubinetto a tre vie e un&#39;estensione Heidelberger per regolare il livello di riempimento e riempire il reattore da 500 ml 0,9% NaCl. <img alt="Figura 2" src="/files/ftp_upload/53029/53029fig2.jpg" /></li></ol> Figura 2:. Schema della perfusione set-up Collegare la camera di perfusione (1.400 cm 3) (1) all&#39;estremità distale (4) del gorgogliatore (5) tramite l&#39;accesso venoso portale (2) o tramite ac arteriosaprocesso (3). Il gorgogliatore (5) deve essere dotato di un otturazione (4) in corrispondenza dell&#39;estremità distale e uno sfiato (6). Collegare il gorgogliatore (5) ad un&#39;estensione Heidelberger (7). Collegare l&#39;estensione Heidelberger al segmento della pompa (8). Inoltre, collegare il segmento della pompa (8) a un altro interno Heidelberger (9). Inserire l&#39;ultima estensione Heidelberger (9) nella bottiglia di soluzione detergente (10). Collegare il respiratore (11) alla camera di perfusione (o al distributore pressione in caso di più perfusioni). Per drenare i fluidi reattore, collegare il deflusso alla bottiglia rifiuti (12). <ol><li> Inserire il segmento pompa nella pompa. Avviare la pompa per riempire il ciclo con PBS, chiudere l&#39;estremità distale del gorgogliatore e aprire la bocchetta. Quando il fondo della bolla è coperto saldamente con PBS (2-3 cm), chiudere lo sfiato e aprire l&#39;estremità distale del gorgogliatore. </li><li> Fegato di installazione <ol><li> Collegare il rubinetto a tre vie della cannula vena porta al relativo accesso reattore. Collegare la cannula arteriosa all&#39;accesso arteriosa. Fare attenzione al fine di garantire che l&#39;aria non entra nei sistemi. </li></ol></li><li> Applicazione di oscillanti Condizioni Pressione <ol><li> Utilizzare un respiratore con una frequenza di 15 bpm e un&#39;ampiezza di 26 mbar (min. 4 mbar, max. 30 mbar). Collegare il respiratore alla camera di distribuzione della pressione e collegare la camera al reattore. Avviare l&#39;applicazione di pressione. </li></ol></li><li> Decellularization Protocol <ol><li> Eseguire passaggi perfusione con un flusso di 5 ml / min. Avviare il decellularization con perfusione del fegato con 1% Triton X-100 per 90 min. Consentire effluence rifiuti tramite il deflusso del dispositivo perfusione per mantenere il livello del fluido all&#39;interno della camera di perfusione e costante del fegato perfuso. Dopo 90 min, cambiare il detergente 1% SDS. <img alt="Figura 3" src="/files/ftp_upload/53029/53029fig3.jpg" /> <img alt="Tabella 1" src="/files/ftp_upload/53029/53029table1.jpg" /></li></ol></li></ol> &lt;p&gt; Figura 3: perfusione Protocollo per tutti i gruppi sperimentali.

<ol>
	<li>Struecker, B., Raschzok, N., Sauer, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+support+strategies:+cutting-edge+technologies.">Liver support strategies: cutting-edge technologies.</a> Nat Rev Gastroenterol Hepatol. 11, 166-176 (2014).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32, 3233-3243 (2011).</li><li>Wang, 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=Decellularized+liver+scaffolds+effectively+support+the+proliferation+and+differentiation+of+mouse+fetal+hepatic+progenitors.">Decellularized liver scaffolds effectively support the proliferation and differentiation of mouse fetal hepatic progenitors.</a> J Biomed Mater Res A. , (2013).</li><li>Uygun, B. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+whole-organ+assembly:+moving+toward+transplantation+on+demand.">Perspectives on whole-organ assembly: moving toward transplantation on demand.</a> J Clin Invest. 122, 3817-3823 (2012).</li><li>Soto-Gutierrez, 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=A+whole-organ+regenerative+medicine+approach+for+liver+replacement.">A whole-organ regenerative medicine approach for liver replacement.</a> Tissue engineering. Part C, Methods. 17, 677-686 (2011).</li><li>De Kock, 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=Simple+and+quick+method+for+whole-liver+decellularization:+a+novel+in+vitro+three-dimensional+bioengineering+tool.">Simple and quick method for whole-liver decellularization: a novel in vitro three-dimensional bioengineering tool.</a> Archives of toxicology. 85, 607-612 (2011).</li><li>Park, K. M., Woo, H. 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The authors have nothing to disclose.

Anche se la tecnica presentata per la raccolta del fegato di ratto e decellularization è facilmente riproducibile, ci sono alcuni passaggi critici da considerare: Durante la preparazione per la raccolta del fegato, è importante evitare emorragia grave perché attiverà la coagulazione del sangue e può portare alla formazione di coaguli di sangue all&#39;interno del fegato. A nostro avviso, è vantaggioso incidere aorta addominale direttamente prima incannulazione della vena porta per evit...

Decellularization e Recellularization possono consentire la generazione di funzionali, organi trapiantabili in vitro 1. By cellule rimozione e materiale antigenico (es., DNA, epitopi alfa-Gal) da un organo, la matrice extracellulare mancato o meno immunogenico (ECM) può essere ottenuto. Questa matrice conserva microanatomia tridimensionale di un organo e può servire come biomatrice ideale per ripopolamento con cellule di un differente, origine eventualmente xenogenico 2. Così, un ratto di matrice fegato decellularized potrebbe essere ripopolata con le cellule di fegato umano. Questo umanizzato micro-fegato potrebbe servire come modello ex vivo per la ricerca sulle malattie (ad es., Le malattie metaboliche congenite, malattie virali o tumori maligni) o per test farmaceutici preclinici 3. Diversi protocolli diversi per fegato di ratto perfusione decellularization sono già stati pubblicati 4-13. In tutti i protocolli, decellularzione è stato ottenuto mediante perfusione di detergenti ionici o non ionici alcaline tramite la vena porta cannulata. Per quanto a nostra conoscenza, siamo stati il primo gruppo a riferire di ratto decellularization fegato perfusione selettiva attraverso la vena porta e / o il ratto arteria epatica 14. Attivazione della perfusione selettiva dei diversi sistemi vascolari nel fegato può consentire migliori risultati Decellularization e, inoltre, può giocare un ruolo importante nel ripopolamento cellulare. Nello studio qui descritto, fegati sono stati perfusi in un dispositivo di perfusione di proprietà su misura, consentendo di perfusione in condizioni di pressione oscillanti. Queste condizioni di pressione imitano la perfusione respiratorie dipendente fisiologica del fegato: in situ, fegato pende sotto la copula del diaframma, il cui movimento durante la respirazione ha un impatto diretto sulla perfusione del fegato. Inspiration conduce specificamente abbassamento del diaframma e spremitura del fegato, ottimizzandoepato-deflusso venoso, che conduce alla scadenza elevazione del fegato e abbassamento della pressione intraddominale ottimizzare afflusso portale-venosa 15. Il nostro obiettivo era quello di valutare se le condizioni di pressione oscillanti hanno un impatto sulla omogeneità del fegato di ratto perfusione decellularization imitando condizioni intraabdominal ex vivo. L&#39;omogeneità del processo decellularization può essere un fattore sottovalutato perfusione decellularization. Tutti gli agenti noti utilizzati per decellularization fegato causa alterazioni della ECM. Le cellule in zone poco irrorate rimangono all&#39;interno della ECM, mentre altre zone sono già completamente decellularized. Per sciogliere le cellule rimanenti, la durata di perfusione o pressione devono essere elevate, causando più alterazioni alle aree ben irrorate-. Così, detergenti per decellularization devono essere distribuiti in modo omogeneo all&#39;interno dell&#39;organo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Self built arterial cannula</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/100</td>
			<td>0,28mm ID 0,61mm OD</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/200</td>
			<td>0,58mm ID 0,96m OD</td>
		</tr>
		<tr>
			<td>Venodrop Safe butterfly catheter</td>
			<td>Fresenius Kabi</td>
			<td>3275851</td>
			<td>21 G</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>portal vein cannula</td>
		</tr>
		<tr>
			<td>Periphereal Venous Catheter</td>
			<td>BD</td>
			<td>393224</td>
			<td>BD Venflon Pro 20G</td>
		</tr>
		<tr>
			<td>three-way stopcock</td>
			<td>smiths medical</td>
			<td>888-101RE</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Cotton Sticks</td>
			<td>Hecht-Assistent</td>
			<td>4302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Pads&#160;</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>13H118-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Bandage</td>
			<td>Hubei Haige&#160; Medical Instruments</td>
			<td>14388</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ringer Solution</td>
			<td>Fresenius Kabi</td>
			<td>13 HKP022</td>
			<td>1000ml</td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td>10ml/ Luer Solo</td>
		</tr>
		<tr>
			<td>5ml Syringe</td>
			<td>Braun</td>
			<td>4606061V</td>
			<td>5ml /Luer Solo</td>
		</tr>
		<tr>
			<td>Suture (Silk 6/0)</td>
			<td>Resorba</td>
			<td>H1F</td>
			<td>LOT 105001.81</td>
		</tr>
		<tr>
			<td>medical drape</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>D0613011</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgical instruments</td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>Geuder</td>
			<td>17570</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>Inox-Electronic</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-scissors</td>
			<td>Martin</td>
			<td>11-740-11</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>S&#38;T&#160;</td>
			<td>112314</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp</td>
			<td>Aesculap</td>
			<td>BH111R</td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>F S T&#160;</td>
			<td>14501-14</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>Aesculap</td>
			<td>BD 557</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Decellularisation</td>
		</tr>
		<tr>
			<td>Respirator</td>
			<td>Resmed</td>
			<td>14.24.11.0004</td>
			<td>SmartAIR ST</td>
		</tr>
		<tr>
			<td>Perfusion Device</td>
			<td>Charite, medical engineering laboratory</td>
			<td>custome-made device</td>
			<td>decellularisation device</td>
		</tr>
		<tr>
			<td>peristaltic pump ismatec reglo ICC</td>
			<td>IDEX</td>
			<td>ISM4408&#160;</td>
			<td>4-channel</td>
		</tr>
		<tr>
			<td>heidelberger extension 75 cm&#160;</td>
			<td>Fresenius Kabi</td>
			<td>2873</td>
			<td>75 cm</td>
		</tr>
		<tr>
			<td>MS/CA pump-segment</td>
			<td>IDEX</td>
			<td>IS 3510&#160;</td>
			<td>MS/CA/click&#39;n&#39;go/POM-C</td>
		</tr>
		<tr>
			<td>CA 2-stopper tube</td>
			<td>Pharmed</td>
			<td>BPT NSF-51</td>
			<td></td>
		</tr>
		<tr>
			<td>bubble trap&#160;</td>
			<td></td>
			<td>custome-made item</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock hose connector</td>
			<td>Neolab</td>
			<td>No. 02-1887</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Detergents</td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth&#160;</td>
			<td>CN30.4&#160;</td>
			<td>2,5 kg</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth&#160;</td>
			<td>3051.1&#160;</td>
			<td>10l</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>DPBS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>staining</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Morphisto</td>
			<td>10177</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer hematoxylin</td>
			<td>AppliChem</td>
			<td>A4840</td>
			<td></td>
		</tr>
		<tr>
			<td>gomori staining</td>
			<td>Morphisto</td>
			<td>11104</td>
			<td></td>
		</tr>
		<tr>
			<td>AlcainBlue-PAS staining</td>
			<td>Morphisto</td>
			<td>11388</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct Red 80&#160;</td>
			<td>Sigma Aldrich</td>
			<td>365548</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for decellularization of rat livers in an oscillating-pressure perfusion device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

L&#39;omogeneità e quindi l&#39;efficacia dei protocolli Decellularization differenti sono stati valutati mediante l&#39;osservazione macroscopica, analisi istologica, e l&#39;analisi del contenuto di DNA rimanendo entro matrici fegato decellularized. Inoltre, la corrosione colata è stata effettuata per visualizzare il microanatomia intatto di fegati dopo decellularization. Macroscopia Durante decellularization, fegati diventano Lucent, che indica la rimozione ...

Watch this Scientific Journal Video about Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device at JoVE.com

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Procedura per Decellularization di Rat Livers in un dispositivo di perfusione oscillante pressione

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

procedure-for-decellularization-rat-livers-an-oscillating-pressure

Research

JoVE Journal

Bioengineering

10.2K Views.  Charité – Universitätsmedizin Berlin. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Video: Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Decellularization e Recellularization di fegati interi

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

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Bioingegneria

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Procedura per decellularizzazione del Cuore di suino da retrograda perfusione coronarica

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Sviluppo di un biosensore elettrochimico del DNA per rilevare un patogeni di origine alimentare

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Due metodi per decellularizzazione di tessuti vegetali per applicazioni di ingegneria del tessuto

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Una nuova tecnica chirurgica come una Fondazione per In Vivo parziale ingegneria del fegato nel ratto

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

Decellularizzazione di cuore umano intero all'interno di una custodia pressurizzata in un orientamento invertito

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Generazione di condizioni ambientali dinamiche utilizzando un dispositivo microfluidico ad alta produttività

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Approvvigionamento e perfusione-decellularizzazione di lembi vascolarizzati suini in un bioreattore di perfusione personalizzato

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Approvvigionamento e decellularizzazione degli arti posteriori di ratto utilizzando un bioreattore ex vivo basato sulla perfusione per allotrapianto composito vascolarizzato

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

Un sistema combinato di micro-dispositivi per promuovere e monitorare la crescita dei coralli

An Integrated Micro-Device System for Coral Growth and Monitoring

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of the coral culture environment is highly in demand for coral conservation and study. Here, we developed a semi-closed coral culture micro-device system as a multi-functional platform, which can provide accurate and programmable temperature control, a sterile initial environment, long-term stable water quality, an adjustable dissolved oxygen concentration, and a customized light spectrum for corals. Owing to the modular design, the coral culture system can be upgraded or modified by installing desirable new modules or removing existing ones. Currently, under appropriate conditions and with proper system maintenance, the sample corals can survive for at least 30 days in a healthy state. Furthermore, due to the controllable and sterile initial environment, this coral culture system can support research into the symbiotic relationship between corals and associated microorganisms. Therefore, this micro-device system can be applied to monitor and investigate sea corals in a relatively quantitative manner.

Autore in primo piano: Avanzamento della coltura dei coralli - Creazione di un sistema di microambiente semi-quantitativamente controllato per contrastare le attuali limitazioni 

This protocol describes the development of a modular controllable micro-device system that can be applied for the long-term culturing and monitoring of sea corals.

Un sistema integrato di micro-dispositivi per la crescita e il monitoraggio dei coralli

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of ...