Gli autori desiderano ringraziare Brogan Guest, Michelle Weaver, e Kristen Lippert. Il finanziamento per questo studio sono state fornite dal NIH Grant R03EB009237, così come le borse di formazione NIH T32EB001026-06 dal National Institute of Biomedical Imaging e Bioingegneria e T32HL076124-05.

1. Preparazione di tessuto ed esperimento di installazione <ol><li> Harvest organo suina immediatamente dopo l&#39;eutanasia da un impianto di macellazione o di ricerca e risciacquare sangue in eccesso. Tagliare il cuore di grasso in eccesso e tessuto, mantenendo gli atri e aorta intatto. Tagliare via il grasso per separare l&#39;arteria polmonare dall&#39;aorta. Se ci sono dei tagli nel tessuto, eliminare in modo appropriato. </li><li> Avvolgere ogni cuore singolarmente in carta freezer e memorizzare tutti i tessuti in un congelatore a -80 ° C per almeno 24 ore per garantire congelamento completo. </li><li> Quando si è pronti per l&#39;uso (di solito meno di 3 mesi), scongelare un cuore intatto suino congelato di tipo 1 acqua per una notte immersi in un bicchiere 4 L a 4 ° C. </li><li> Dopo che il cuore è completamente scongelato, accarezzare il cuore arido, pesare il cuore, e registrare il peso. Il cuore di un maiale del peso di mercato dovrebbe pesare circa 375-450 g. </li><li> Collegare taglia 18 tubo Masterflex alla fine ¼ &quot;di un riduttore spinato. Inserire il riduttore spinato etubo dentro l&#39;aorta. Luogo 2 fascette o fascette sicure in tutto il aorta, appena sotto il tronco brachiocefalico. Il riduttore e la tubazione deve rimanere al di sopra della valvola aortica, così le arterie coronarie possono essere perfuso (Figura 1). </li><li> Utilizzare una siringa da 30 o 60 ml per riempire il tubo con acqua di tipo I. Inserire il tubo all&#39;interno della cartuccia di una pompa a rulli Masterflex nel suo punto medio approssimativo. Immergere l&#39;estremità del tubo di afflusso nel fondo di un bicchiere 4 L riempito con 2,5 L di acqua e fissare il tubo. </li><li> Posizionare il cuore nel becher riempito con acqua, e adescare la pompa per rimuovere le bolle d&#39;aria. Se si osservano bolle proveniente dalla aorta dove è inserito il tubo, l&#39;aorta può essere necessario riposizionare o fissato con legami aggiuntivi. Una chiusura ermetica è importante per mantenere la pressione necessaria durante il processo di decellularizzazione (Figura 2). </li><li> Posizionare i 4 becher contenente L 3 L di un 0,2% Trypsin/0.05% EDTA/0.05% NaN3 soluzione sul piatto mescolare e riscaldare a 37 ° C, in preparazione del processo di decellularizzazione. </li></ol> 2. Tissue Risciacqui <ol><li> Impostare la pompa ad una portata di 400 ml / min, assicurando che la misura del tubo sia selezionata. Lavare il cuore di tipo I acqua per 15-25 minuti. Poiché la pompa viene avviata, il cuore deve gonfiarsi e effonderà sangue dai ventricoli. Soluzione fresca dovrebbe essere sostituito ogni 5-10 minuti, o quando necessario sulla base della quantità di sangue prelevato dal cuore. Se il sangue non è effuso dal cuore, regolare il tubo e morsetti sufficiente. </li><li> Arrestare la pompa e trasferire il cuore in un becher separato riempito di 2X Phosphate Buffered Saline (PBS). Dopo il tubo viene immerso in soluzione, avviare la pompa e aumentare il flusso di 700 ml / min. Il cuore dovrebbe rimanere in soluzione per 15 minuti, cambiando la soluzione ogni 5 min. Ogni cambiamento soluzione richiede la pompa da arrestare temporaneamente mentre il tessuto e il tubo viene spostatonel becher nuovo. </li><li> Trasferire il cuore di tipo I di acqua per 10 min e aumentare il flusso di 750 ml / min. </li></ol> 3. Decellularizzazione e soluzione di perfusione <ol><li> Trasferire il cuore nel becher contenente 0,2% Trypsin/0.05% EDTA/0.05% NaN 3 a 37 ° C. Aumentare la velocità della pompa a 1.200 ml / min e avviare la pompa. Utilizzare un agitatore posizionato sul fondo del bicchiere per far circolare la soluzione nel becher. Il cuore deve rimanere nella Trypsin/0.05% 0,2% EDTA/0.05% NaN 3 soluzione a 37 ° C per un totale di tre ore. Dopo 1 h, aumentare la velocità della pompa a 1.500 ml / min. Dopo un&#39;altra ora, aumentare la velocità della pompa a 1800 ml / min. Il tessuto è sottoposto a lenta velocità di perfusione aumento alla condizione del tessuto e prevenire la rottura dei vasi. Il cuore si gonfiano e quasi raddoppia durante questa fase del protocollo. Il tessuto perderà il suo colore naturale, procedendo dagli atri ai all&#39;apice tutto °e protocollo (Figura 3). </li><li> Dopo ogni soluzione di perfusione, in due fasi di risciacquo viene eseguita per rimuovere i detriti cellulari, residui chimici, e lisi cellulare aiuto. Ogni lavaggio è costituito da un risciacquo 10 min in acqua di tipo I seguita da 10 min risciacquo con soluzione 2X PBS a temperatura ambiente. Ogni lavaggio consiste nella rimozione della soluzione dal recipiente originale, aggiungere soluzioni di risciacquo, e la circolazione del perfusato nel becher contenente il cuore sommerso. Dopo il 0,2% Trypsin/0.05% EDTA/0.05% NaN 3 soluzione, acqua profumato a 1.900 ml / min e quindi perfondere 2X PBS a 1950 ml / min. </li><li> Trasferire il cuore ad una soluzione di 3% Triton X-100/0.05% EDTA/0.05% NaN 3 a temperatura ambiente. Aumentare la velocità della pompa a 2.000 ml / min e profumato soluzione per un&#39;ora. Rimuovere la soluzione dal bicchiere e sostituire con soluzione fresca, aumentare la velocità della pompa a 2100 ml / min, e perfondere la soluzione fresca per un&#39;altra ora e mezzo, portando il tempo totale in3% Triton X-100/0.05% EDTA/0.05% NaN 3-2,5 ore. </li><li> Risciacquare il tessuto in acqua di tipo I a 2150 ml / min e 2X PBS a 2180 ml / min per 10 min. </li><li> Trasferire il cuore di una soluzione di sodio desossicolato 4% a temperatura ambiente. Aumentare la velocità della pompa a 2200 ml / min e profumato soluzione per 3 ore. </li><li> Risciacquare il tessuto in acqua a tipo I e 2X PBS a 2.200 ml / min per 15 min ciascuna, cambiando le soluzioni dopo 5-10 minuti per ogni soluzione. Le fasi di perfusione descritti possono essere suddivisi su più giorni eseguendo la fase di lavaggio due volte e memorizzazione del cuore con tubo attaccato notte a 4 ° C e sommersa in acqua Tipo I. </li><li> Il giorno seguente, eseguire un 5 min risciacquo con acqua di tipo I a 750 ml / min, seguito da un risciacquo 5 min in 1X PBS a 1.500 ml / min. Il protocollo può essere continuato con la portata descritto nella soluzione adeguata. </li></ol> 4. Disinfezione e trattamento finale <ol><li> Trasferire il cuore ad un Perac 0,1%ETIC acido / soluzione di etanolo al 4% e la soluzione profumato per 1,5 ore a 2.200 ml / min. </li><li> I risciacqui finali per i tessuti sono tutti eseguiti in 2.200 ml / min. Perfondere il tessuto con 1X PBS per 15 minuti, seguito da due lavaggi 5 min in acqua di tipo I. Questa serie di risciacqui viene ripetuta ancora una volta, per completare la procedura di perfusione soluzione. </li><li> Spegnere la pompa e rimuovere il cuore da soluzione di scendere al cuore. Tagliare i legami della aorta, rimuovere tutti i tubi, e posizionare il cuore in un bicchiere vuoto a sgocciolare per 1 ora. Liquido in eccesso deve essere svuotato periodicamente. Gettare il cuore su un cuscinetto assorbente per drenare completamente il cuore (Figura 4). </li><li> Dopo la maggior parte dell&#39;acqua viene rimossa, registrare il peso della matrice extracellulare cardiaca (C-ECM). Il cuore può aspettare di perdere circa il 20-25% del suo peso iniziale durante il processo di decellularizzazione. </li><li> Sezionare i ventricoli destro e sinistro, così come il setto ventricolare per DNA quantificatie il trattamento istologico per confermare decellularizzazione completa del tessuto (Figura 5). </li><li> Congelare il C-ECM a -80 ° C per almeno 2 ore prima della liofilizzazione. </li></ol>

<ol>
	<li>Ott, H. 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=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat. Med. 16, 927-933 (2010).</li><li>Ott, H. 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=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat. Med. , (2008).</li><li>Petersen, T. 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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=Strategies+for+tissue+and+organ+decellularization.">Strategies for tissue and organ decellularization.</a> Journal of cellular biochemistry. , (2012).</li><li>Gilbert, T. W., Sellaro, T. L., 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=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3683 (2006).</li><li>Akhyari, P., 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+quest+for+an+optimized+protocol+for+whole-heart+decellularization:+a+comparison+of+three+popular+and+a+novel+decellularization+technique+and+their+diverse+effects+on+crucial+extracellular+matrix+qualities.">The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities.</a> Tissue Eng. Part C Methods. 17, 915-926 (2011).</li><li>Weymann, 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=Development+and+evaluation+of+a+perfusion+decellularization+porcine+heart+model--generation+of+3-dimensional+myocardial+neoscaffolds.">Development and evaluation of a perfusion decellularization porcine heart model--generation of 3-dimensional myocardial neoscaffolds.</a> Circulation journal : official journal of the Japanese Circulation Society. 75, 852-860 (2011).</li><li>Cortiella, 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=Influence+of+acellular+natural+lung+matrix+on+murine+embryonic+stem+cell+differentiation+and+tissue+formation.">Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.</a> Tissue Eng. Part A. 16, 2565-2580 (1089).</li><li>Remlinger, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrated+xenogeneic+decellularized+tracheal+matrix+as+a+scaffold+for+tracheal+reconstruction.">Hydrated xenogeneic decellularized tracheal matrix as a scaffold for tracheal reconstruction.</a> Biomaterials. 31, 3520-3526 (2010).</li><li>Sellaro, T. L., Ravindra, A. K., Stolz, D. B., 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=Maintenance+of+hepatic+sinusoidal+endothelial+cell+phenotype+in+vitro+using+organ-specific+extracellular+matrix+scaffolds.">Maintenance of hepatic sinusoidal endothelial cell phenotype in vitro using organ-specific extracellular matrix scaffolds.</a> Tissue Eng. 13, 2301-2310 (2007).</li><li>Wainwright, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+ventricular+outflow+tract+repair+with+a+cardiac+biologic+scaffold.">Right ventricular outflow tract repair with a cardiac biologic scaffold.</a> Cells, tissues, organs. 195, 159-170 (2012).</li></ol>

Il dottor Gilbert era sul Scientific Advisory Board a Acell, Inc. mentre lo studio era stato fatto, e recentemente è diventato il VP di Ricerca e Sviluppo. Acell, Inc. vende matrice della vescica urinaria e non ha alcun interesse commerciale in questo studio.

Lo studio attuale metodologia descritta per decellularizzazione coerente ed efficace di un cuore suino. Il protocollo era una modifica a un precedentemente pubblicato con il rapporto 1, e comprendeva più l&#39;esposizione al flusso e aumento della pressione, che ha fornito risultati più ripetibili. Il tessuto risultante decellularizzato soddisfatto tutti i criteri pubblicati per decellularizzazione successo del tessuto 2. Soluzione cambia frequenti sono stati eseguiti per limitare la reintroduzio...

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Tripsina </td><td> Gibco </td><td> 15090 </td><td></td></tr><tr><td> EDTA </td><td> Pescatore </td><td> BP120-500 </td><td></td></tr><tr><td> NaN 3 </td><td> Sigma </td><td> S2002-500G </td><td></td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> X100-1L </td><td></td></tr><tr><td> 10X PBS </td><td> Pescatore </td><td> BP399-20 </td><td></td></tr><tr><td> Sodio desossicolato </td><td> Sigma </td><td> D6750-500G </td><td></td></tr><tr><td> Acido peracetico </td><td> Pfaltz e Bauer </td><td> P05020 </td><td> 35% CAS # 79-21-0 </td></tr><tr><td> Etanolo </td><td> Pharmco </td><td> 111000200 </td><td></td&gt; </tr><tr><td> Masterflex azionamento della pompa </td><td> Cole Parmer </td><td> SI-07.524-50 </td><td></td></tr><tr><td> Masterflex Tubi </td><td> Cole Parmer </td><td> 96400-18 </td><td> Size 18 </td></tr><tr><td> Riduttore spinato </td><td> Cole Parmer </td><td> EW-30612-20 </td><td></td></tr><tr><td> Beaker 4L </td><td> Fisher Scientific </td><td> 02-540T </td><td></td></tr></tbody></table>

procedure for decellularization of porcine heart by retrograde coronary perfusion

Perfusione basata decellularizzazione intero organo ha recentemente guadagnato interesse nel campo dell&#39;ingegneria dei tessuti come mezzo per creare sito-specifici scaffold della matrice extracellulare, mentre gran parte conservando l&#39;architettura nativa del ponteggio. Ad oggi, questo approccio è stato utilizzato in una varietà di sistemi di organi, compreso il cuore, polmone, fegato e 1-5. Decellularizzazione metodi precedenti per tessuti senza una rete facilmente accessibile vascolare hanno invocato esposizione prolungata del tessuto a soluzioni di detergenti, acidi, o trattamenti enzimatici come mezzo per rimuovere i componenti cellulari e nucleari dall&#39;ambiente circostante extracellulare 6-8. Tuttavia, l&#39;efficacia di questi metodi incernierato sulla capacità delle soluzioni di permeare il tessuto tramite diffusione. In contrasto, la perfusione di organi attraverso il sistema vascolare naturale ridotto efficacemente la distanza di diffusione e trasporto facilitato di decellularizatisugli agenti nel tessuto e componenti cellulari dal tessuto. Qui, si descrive un metodo per decellularize pienamente un cuore intatto suino attraverso perfusione coronarica retrograda. Il protocollo ha prodotto completamente decellularizzato cardiaca matrice extracellulare (ECM-c) scaffold con la struttura tridimensionale del cuore intatto. Il nostro metodo utilizzato una serie di enzimi, detergenti e acidi accoppiato con risciacqui ipertoniche e ipotoniche per facilitare la lisi delle cellule e la rimozione. Il protocollo utilizzato una soluzione di tripsina per staccare celle della matrice seguito da Triton X-100 e sodio desossicolato soluzioni per aiutare nella rimozione di materiale cellulare. Il protocollo descritto utilizza anche velocità di perfusione maggiore di 2 L / min per lunghi periodi di tempo. L&#39;elevata portata, accoppiato con soluzione cambia accettati trasporto di agenti al tessuto senza contaminazione di detriti cellulari e assicurato efficace risciacquo del tessuto. Il metodo descritto rimosso tutto il materiale nucleare da native suina tessuto cardiaco, creando un sito-specifico cardiaca ECM scaffold che può essere utilizzato per una varietà di applicazioni.

L&#39;effetto di decellularizzazione sui cuori suina tutto varia naturalmente a causa delle differenze in termini di dimensioni, pressioni, e la disposizione del serbatoio. Pertanto, la composizione esatta degli scaffold derivati ​​matrice extracellulare non sarà lo stesso da cuore a cuore. Il completamento del protocollo descritto produrrà un cuore che appare bianco o traslucido, indicando la perdita di materiale cellulare. Tuttavia, è ampiamente accettato che un tessuto può essere considerata &quot;decellulari...

Watch this Scientific Journal Video about Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion at JoVE.com

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Un metodo per rimuovere rapidamente e completamente componenti cellulari da un cuore suino intatta attraverso perfusione retrograda viene descritto. Questo metodo fornisce uno specifico sito cardiaca matrice extracellulare ponteggio che ha il potenziale per l&#39;utilizzo in molteplici applicazioni cliniche.

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 ...

procedure-for-decellularization-porcine-heart-retrograde-coronary

Research

JoVE Journal

Bioengineering

23.7K Views.  McGowan Institute for Regenerative Medicine. Un metodo per rimuovere rapidamente e completamente componenti cellulari da un cuore suino intatta attraverso perfusione retrograda viene descritto. Questo metodo fornisce uno specifico sito cardiaca matrice extracellulare ponteggio che ha il potenziale per l'utilizzo in molteplici applicazioni cliniche.

Video: Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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 ...

Ricerca

Bioingegneria

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.

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 ...

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 ...

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

Isolamento e decellularizzazione di un Pancreas suino intero

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Rilevamento dell'endotossina in Nano-formulazioni utilizzando dosaggi di Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

Misurazione in vivo risolta nel tempo della dinamica dei neuropeptidi mediante immunosonda capacitiva nel cuore suino

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

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 ...

Valutazione delle proprietà idrodinamiche nelle valvole aortiche suine montate

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

Autore in primo piano: Avanzamento dei test sulle procedure cardiache prima di intraprendere studi su animali di grossa taglia 

We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model.

Un modello suino ex vivo per test idrodinamici di procedure sperimentali di valvole aortiche e nuovi dispositivi medici

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...