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

Les animaux ont été maintenus à la Facilité pour la médecine expérimentale (FEM, Charité, Berlin, Allemagne), et tous les protocoles expérimentaux ont été examinées et approuvées par l&#39;Office d&#39;Etat de la Santé et des Affaires locales (LAGeSo, Berlin, Allemagne;. Reg n ° O 0365 / 11). 1. Foie récolte <ol><li> Préparation pré-opératoire <ol><li> Utilisez une plaque de liège pour la fixation. Mettez un drap médicale sur la plaque. En utilisant quatre aiguilles, fixer un masque d&#39;inhalation sur la plaque de liège pour peropératoire inhalation narcose. </li></ol></li><li> Prédisposition du Portail veineuse canule <ol><li> Connecter un cathéter veineux périphérique (G 20) pour un robinet d&#39;arrêt à trois voies. Cathéter veineux doit être coupé obliquement, avec une longueur d&#39;env. 1,5 cm. </li><li> De l&#39;air le système en utilisant une seringue de 10 ml remplie de solution de Ringer. Il est très important de bien désaérer parce embolie gazeuse peut affecter négativement la décellularisation de perfusion. </li></ol></li><li> Prédispositionde la canule artérielle <ol><li> Utilisez une canule papillon et couper l&#39;aiguille à une longueur de 5 mm. Monter un tube de 5 cm (ID 0,58 mm; OD 0,96 mm) à l&#39;aiguille de 5 mm et la fixer avec de la colle. Insérez un tube de 2 cm de long (ID 0,28 mm; OD 0,61 mm) dans le plus grand tube et le fixer avec de la colle. </li><li> Glissez un autre tube sur cette construction comme une butée et le fixer avec de la colle. De l&#39;air de la canule artérielle avec une seringue de 5 ml rempli d&#39;une solution de Ringer. </li></ol></li><li> Anesthésie et l&#39;analgésie <ol><li> Induire une narcose du rat dans la chambre d&#39;induction de l&#39;anesthésie par 3,5% d&#39;isoflurane dans de l&#39;oxygène à un débit de 1,5 L / min. </li><li> Pour assurer une analgésie sécuritaire pendant l&#39;opération, injecter metamizol (100 mg / kg de masse corporelle) et la kétamine à faible dose / médétomidine (10 mg / 0,1 mg par kg de masse corporelle) par voie intrapéritonéale. </li><li> Fournir 1,5% d&#39;isoflurane dans 1 L / min d&#39;oxygène via le masque d&#39;inhalation pour l&#39;entretien ultérieur de l&#39;anesthésie chirurgicale. </li></ol></li><li> Arrangements préopératoires &lt;ol&gt; <li> Raser l&#39;abdomen généreusement avec une cisaille électrique. Humidifier le site de l&#39;incision de l&#39;abdomen rasé de l&#39;éthanol à 70% pour la désinfection. </li><li> Placez l&#39;animal dans la position couchée sur la plaque préparée, insérer la tête dans le masque d&#39;inhalation et de fixer les branches avec du ruban adhésif médical et broches. Enlever l&#39;excès de l&#39;éthanol et de la fourrure avec une compresse de gaze. </li></ol></li><li> Évaluation de l&#39;anesthésie <ol><li> Évaluer la profondeur de l&#39;anesthésie en observant la fréquence respiratoire. Lorsque l&#39;anesthésie semble assez profond (40-60 respirations / min), vérifier si l&#39;analgésie est suffisante en essayant de déclencher le réflexe orteil pincée aide de pinces chirurgicales de pincer la peau entre les orteils des deux membres postérieurs. </li></ol></li><li> Approche abdominale <ol><li> Faire une incision médiane dans la paroi abdominale caudale dessus de la vessie. Couper de ce point à deux arches latérales côtières dans une forme de V. Prenez soin de ne pas couper dans le diaphragme. Tirez sur la paroi abdominale couper sur la poitrine. </li&gt; <li> Fixer la pointe du sternum avec une pince de Overholt, tirez-crânienne et le fixer avec une bande de caoutchouc. Utilisez des cotons-tiges humides d&#39;évacuer l&#39;intestin. Enveloppez l&#39;intestin dans un bandage de gaze humide. En outre, couvrir toutes les marges abdominaux avec une compresse humide. </li></ol></li><li> Préparation du foie <ol><li> Disséquer le ligament falciforme. Utilisez un pansement de gaze humide pour maintenir les lobes du foie médiales et gauche crânialement sous le dôme du diaphragme. Exposer le pédicule hépatique et l&#39;omental lobe du foie supérieure. </li><li> Utilisez deux microforceps et ciseaux à ressort à disséquer soigneusement le ligament attaché au lobe omental supérieure jusqu&#39;à ce que le lobe est mobile. Déplacez le lobe utilisant des cotons-tiges humides sous la compresse de gaze, tenant les lobes médian et gauche en place. </li><li> Après la mobilisation de l&#39;omental lobe hépatique supérieure, déplacer l&#39;estomac vers la gauche. Fixer l&#39;estomac avec une pince et de disséquer l&#39;épiploon mineur avec microforceps et les ciseaux à ressort. </li><li> Mobiliser l&#39;omenta inférieurel de lobe de foie jusqu&#39;à ce qu&#39;il est mobile et se déplacer ensuite le lobe de retour dans sa cavité. Utilisez une pince Backhaus de conserver le duodénum. Déplacez le duodénum vers la gauche pour étirer et exposer le pédicule hépatique. Circonscrire le canal cholédoque dans le ligament hépato étiré. </li><li> On dissèque la voie biliaire principale du tissu adipeux et du pancréas. Couper le canal biliaire 1,5 cm de la bifurcation de la voie biliaire. Disséquer la veine porte du tissu adipeux environnant. Exposer la veine gastro-duodénale. </li><li> Ligaturer la veine gastro deux fois avec de la soie 6-0 et diviser la veine entre les deux ligatures. Disséquer le tronc coeliaque et l&#39;artère hépatique commune du tissu environnant jusqu&#39;à ce que tous rami artérielle sont révélés. </li><li> Libérer l&#39;artère gastro-duodénale du tissu environnant. Attachez l&#39;artère gastro-duodénale deux fois avec de la soie 6-0, avec les liens éloignés les uns des autres. Disséquer l&#39;artère gastro-duodénale entre les deux ligatures. </li><li> Libérer l&#39;artère spléniquedu tissu environnant. Attachez l&#39;artère splénique deux fois avec de la soie 6-0, avec les liens éloignées l&#39;une de l&#39;autre. Disséquer l&#39;artère splénique entre les deux ligatures. </li><li> Libérer l&#39;artère gastrique gauche du tissu environnant. Attachez l&#39;artère gastrique gauche deux fois avec de la soie 6-0, avec les liens éloignés les uns des autres. Disséquer l&#39;artère entre les deux ligatures. </li><li> Circonscrire la veine cave inférieure entre le rein droit et le lobe droit du foie latérale. Disséquer la veine de l&#39;espace rétro-péritonéal. Exposer l&#39;aorte en suivant le tronc cœliaque. </li><li> Disséquer le tissu adipeux rétropéritonéale. Injecter 1,000 IE héparine dans 1 ml de sérum physiologique dans la veine cave inférieure. Rétracter la canule et fermer l&#39;incision avec un tampon de coton. Attendez 1-2 min de l&#39;effet systémique de l&#39;héparine. </li><li> Circonscrire l&#39;aorte avec une pince. Disséquer l&#39;aorte et être exsangue le rat. En outre, disséquer la veine cave inférieure de manière distale depuis le rein. </li></ol></li><li> Portail veineuse Canulation (utilisation de la canule préparé dans la section 1.2) <ol><li> Faire une incision poissons-bouche dans la veine distale. Ouvrez l&#39;incision avec une pince et cathétériser la veine porte. Rincez le foie avec 20 ml de solution de Ringer jusqu&#39;à ce que les décolore hépatiques et fixer la canule avec ligatures (soie 6-0). </li></ol></li><li> Arterial Canulation (utilisation de la canule préparé dans la section 1.3) <ol><li> Disséquer l&#39;aorte au-dessus du tronc cœliaque. Libérer le segment de l&#39;aorte de l&#39;espace rétro-péritonéal. Couper le segment de l&#39;aorte longitudinalement pour générer un correctif aortique. Insérez la canule construit soi-même dans un support statique. </li><li> Utilisez 2 pinces à glisser le patch aortique au cours de la canule. Ligaturer l&#39;artère hépatique avec de la soie 6-0 sur la canule et fixer le patch à la butée. </li></ol></li><li> Foie explantation <ol><li> Ouvrez le diaphragme, faire une incision circulaire et de disséquer le foie contre les structures environnantes. </li></ol></li><li> Stockage <ol><li> Store le foie dans un réservoir de 500 ml stérile rempli de 350 ml de solution de Ringer jusqu&#39;à utilisation. </li></ol></li></ol> 2. détergents Solutions <ol><li> Solution à 10% de Triton X-100 actions <ol><li> Remplissez une bouteille de 1000 ml avec 900 ml d&#39;eau distillée. et ajouter 100 ml de Triton X-100 (99,9%). Utilisation d&#39;un agitateur pour dissoudre le Triton X-100. Remplir la bouteille avec de l&#39;eau distillée. jusqu&#39;à un volume total de 1000 ml soit atteinte. </li></ol></li><li> 1% de Triton X-100 <ol><li> Ajouter 100 ml de la solution mère (10%) pour une bouteille de 1000 ml. Ajouter 900 ml d&#39;eau distillée. et secouer deux fois la bouteille. Filtrer la solution à 1% de Triton X-100 à travers un filtre stérile. </li></ol></li><li> 10% de SDS Stock Solution <ol><li> Remplissez une bouteille de 1000 ml avec 900 ml d&#39;eau distillée. et d&#39;ajouter 66,99 g SDS (99,9%, la densité de 0,67). Utiliser un agitateur pour dissoudre le SDS. Remplir la bouteille avec de l&#39;eau distillée. jusqu&#39;à un volume total de 1000 ml soit atteinte. </li></ol></li><li> 1% de SDS <ol><li> Ajouter 100 ml de la solution mère (10%) à un 1000 ml botTLE. Ajouter 900 ml d&#39;eau distillée. et secouer deux fois la bouteille. Filtrer la solution à 1% de SDS à travers un filtre stérile. </li></ol></li></ol> 3. Décellularisation <ol><li> Set-up de perfusion périphérique <img alt="Figure 1" src="/files/ftp_upload/53029/53029fig1.jpg" /></li></ol> Figure 1:. Schéma de l&#39;appareil de perfusion pour l&#39;artériopathie sélective et Portal veineuse perfusion des Quatre Rat Livers sous oscillants Conditions de pression (. Modifié depuis Struecker et al 14) <ol><li> Relier le drain à un robinet à trois voies et une extension Heidelberger pour réguler le niveau de remplissage et remplir le réacteur avec 500 ml de NaCl 0,9%. <img alt="Figure 2" src="/files/ftp_upload/53029/53029fig2.jpg" /></li></ol> Figure 2:. Schéma de la perfusion set-up Lier la chambre de perfusion (1400 cm 3) (1) à l&#39;extrémité distale (4) du piège à bulles (5) via l&#39;accès veineux portal (2) ou via l&#39;ac artérielleprocessus (3). Le piège à bulles (5) doit être équipé d&#39;une obturation (4) à l&#39;extrémité distale et un évent (6). Branchez le piège à bulles (5) à une extension Heidelberger (7). Liez l&#39;extension Heidelberger au segment de pompe (8). En outre, connecter le segment de pompe (8) à un autre poste Heidelberger (9). Insérez la dernière prorogation Heidelberger (9) dans le flacon de solution de détergent (10). Connecter le respirateur (11) à la chambre de perfusion (ou de la distribution de pression dans le cas des perfusions multiples). Pour drainer les fluides de réacteurs, connectez la sortie à la bouteille de déchets (12). <ol><li> Insérez le segment de pompe dans la pompe. Démarrer la pompe pour combler le cycle avec du PBS, fermer l&#39;extrémité distale de la piège à bulles et d&#39;ouvrir l&#39;évent. Lorsque le fond de la bulle est bien couvert avec du PBS (2-3 cm), fermez l&#39;évent et ouvrir l&#39;extrémité distale de la piège à bulles. </li><li> Foie Installation <ol><li> Liez le robinet à trois voies de la canule de la veine à l&#39;accès du réacteur connexe. Connect la canule artérielle à l&#39;accès artériel. Veillez à ce que l&#39;air ne pénètre dans les systèmes. </li></ol></li><li> Application de oscillantes Conditions de pression <ol><li> Utiliser un respirateur avec une fréquence de 15 battements par minute et une amplitude de 26 mbar (min. 4 mbar, max. 30 mbar). Connectez le respirateur à la chambre de répartition de la pression et de relier la chambre du réacteur. Lancement de l&#39;application de la pression. </li></ol></li><li> Protocole de Décellularisation <ol><li> Effectuer les étapes de perfusion avec un débit de 5 ml / min de débit. Lancer la décellularisation de la perfusion du foie avec 1% de Triton X-100 pendant 90 min. Autoriser déchets effluents via la sortie du dispositif de perfusion pour maintenir le niveau du liquide à l&#39;intérieur de la constante de la chambre de perfusion et du foie perfusé. Après 90 min, changer le détergent à 1% de SDS. <img alt="Figure 3" src="/files/ftp_upload/53029/53029fig3.jpg" /> <img alt="Tableau 1" src="/files/ftp_upload/53029/53029table1.jpg" /></li></ol></li></ol> &lt;p&gt; Figure 3: Protocole de perfusion pour tous les groupes expérimentaux.

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The authors have nothing to disclose.

Bien que la technique présentée pour la récolte de foie de rat et décellularisation est facilement reproductible, il ya certaines étapes critiques à prendre en considération: Lors de la préparation pour la récolte du foie, il est important d&#39;éviter une hémorragie sévère, car il va activer la coagulation du sang et peut conduire à la formation d&#39;un caillot sanguin dans le foie. À notre avis, il est avantageux d&#39;inciser l&#39;aorte abdominale directement devant une c...

Décellularisation et recellularization peuvent permettre la génération d&#39;organes transplantables, fonctionnels in vitro 1. En éliminant les cellules et du matériel antigénique (par ex., ADN, des epitopes a-Gal) à partir d&#39;un organe, la matrice extracellulaire non immunogène ou moins (ECM) peut être obtenu. Cette matrice conserve la microanatomie trois dimensions d&#39;un organe et peut servir de biomatrice idéal pour le repeuplement de cellules d&#39;une autre, l&#39;origine xénogénique peut-être deux. Ainsi, une matrice de foie de rat décellularisé peut être repeuplée avec des cellules de foie humain. Ce micro-foie humanisé pourrait servir de modèle ex vivo pour la recherche sur les maladies (par exemple, des maladies métaboliques. Innées, les maladies virales ou malignes) ou pour les tests pharmaceutique préclinique 3. Plusieurs protocoles différents pour le foie de rat perfusion décellularisation ont déjà été publiés 4-13. Dans tous les protocoles, decellularsation a été réalisée par perfusion de détergents ioniques ou non ioniques alcalins via la veine canulée. Pour le meilleur de nos connaissances, nous étions le premier groupe à signaler rat décellularisation du foie par perfusion sélective via la veine porte et / ou le rat de l&#39;artère hépatique 14. Activation de la perfusion sélective des différents systèmes vasculaires dans le foie peut permettre de meilleurs résultats de décellularisation et, en outre, peut jouer un rôle important dans repeuplement cellulaire. Dans l&#39;étude détaillée ici, foies ont été perfusés dans un dispositif de perfusion exclusive sur mesure, permettant perfusion dans des conditions de pression oscillants. Ces conditions de pression imitent la perfusion respiratoire dépendant physiologique du foie: in situ, le foie se suspend sous la copule de la membrane, dont le mouvement lors de la respiration a un impact direct sur ​​la perfusion du foie. Inspiration conduit spécifiquement à l&#39;abaissement du diaphragme et pressant du foie, en optimisanthépato-veineuse sortie, tandis expiration conduit à l&#39;élévation du foie et de l&#39;abaissement de la pression intra-abdominale d&#39;optimiser afflux portail 15-veineuse. Notre objectif était d&#39;évaluer si oscillant conditions de pression ont un impact sur ​​l&#39;homogénéité de foie de rat perfusion décellularisation en imitant les conditions intra-abdominales ex vivo. L&#39;homogénéité du processus de décellularisation peut être un facteur sous-estimé dans la perfusion décellularisation. Tous les agents connus utilisés pour décellularisation de provoquer des altérations du foie à l&#39;ECM. Les cellules dans les zones mal perfusés restent dans l&#39;ECM, alors que d&#39;autres zones sont déjà complètement décellularisée. Pour dissoudre les cellules restantes, la durée de perfusion ou de pression doivent être élevés, causant plus de modifications dans les zones bien irrigués. Ainsi, les détergents pour décellularisation devraient être distribués de manière homogène au sein de l&#39;organe.

<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;homogénéité et donc l&#39;efficacité des différents protocoles de décellularisation ont été évalués par observation macroscopique, l&#39;analyse histologique et l&#39;analyse de la teneur en ADN restant à l&#39;intérieur des matrices de foie décellularisés. En outre, la corrosion coulée a été effectuée pour visualiser le microanatomie intacte du foie après décellularisation. Macroscopie Pendant décellularisation, foies deviennent lucent...

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.

Procédure pour Décellularisation du Rat Livers dans une perfusion périphérique oscillant de pression

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 vues.  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.

Vidéo: 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.

Décellularisation et Recellularization des foies entiers

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 ...

Recherche

Bio-ingénierie

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.

Procédure de décellularisation de coeur de porc par perfusion coronaire rétrograde

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

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

Un outil à ultrasons pour la Conduction nerveuse bloquer dans les modèles de Rat diabétique

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

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.

Développement d’un Biocapteur électrochimique ADN pour détecter un pathogène d’origine alimentaire

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

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.

Une nouvelle Technique chirurgicale comme fondement pour In Vivo partielle génie hépatique chez le Rat

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.

DÉCELLULARISATION du cœur humain à l’intérieur d’une poche sous pression dans une Orientation inversée

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.

Génération de conditions environnementales dynamiques à l’aide d’un dispositif microfluidique à haut débit

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.

Acquisition et perfusion-décellularisation de lambeaux vascularisés porcins dans un bioréacteur de perfusion personnalisé

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.

Obtention et décellularisation de membres postérieurs de rat à l’aide d’un bioréacteur ex vivo basé sur la perfusion pour l’allotransplantation composite vascularisée

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

Un système combiné de micro-dispositifs pour favoriser et surveiller la croissance des coraux

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.

Pleins feux sur l’auteur : Faire progresser la culture des coraux - Créer un système de microenvironnement semi-contrôlé quantitativement pour contrer les limites actuelles 

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 système de micro-dispositifs intégré pour la croissance et la surveillance des coraux

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