Les auteurs tiennent à remercier Jack Milwid pour la conception de la perfusion in vitro dans la chambre. Ce travail a été soutenu par des subventions du NIH américain, R01DK59766 et R01DK084053 à MY, R00DK080942 au KU, US NSF CBET-0853569 au KU et les Hôpitaux Shriners pour enfants au BEU (subvention aucune. 8503). Nous reconnaissons aussi le soutien et les Hôpitaux Shriners pour enfants.

1. Décellularisation foie <ol><li> Récolte d&#39;un foie de rat avec canulation veine porte et de l&#39;aide de calibre 18 cathéter. Laisser l&#39;inférieur et la veine cave supérieure ouverte. Gardez l&#39;organe hydraté dans du tampon phosphate salin (PBS) dans un plat de 10 cm de Pétri. </li><li> Mettre en place un système de perfusion qui se compose de réservoir de 8 litres, pompe péristaltique et un piège à bulles. </li><li> Remplir le système de perfusion avec une solution saline tamponnée de phosphate et le faire fonctionner pendant 10 minutes. Remplissez PBS dans un plat de 10 cm de Pétri et réduire le débit de tampon phosphate salin à 1 mL / min. </li><li> Transférer soigneusement le foie récoltés à l&#39;tampon phosphate salin remplie de 10 cm boîte de Pétri. </li><li> Continuez avec une perfusion PBS pendant une nuit. </li><li> Démarrer la perfusion avec 0,01% (p / v) de dodécylsulfate de sodium (SDS) en eau distillée pendant 5 minutes. </li><li> Perfuser avec du PBS pendant 1 heure. </li><li> Répétez les étapes 1.6 et 1.7 plus de trois fois l&#39;augmentation du temps de perfusion SDS à 10 minutes, 15 et 20 à chaque fois. </li><li> Poursuivre la perfusion avec 0,01% (p / v) de SDS pendant 24 heures. </li><li> Poursuivre la perfusion avec 0,1% (p / v) de SDS pendant 24 heures. </li><li> Perfuser avec 0,2% (p / v) SDS pendant 3 heures. </li><li> Perfuser avec 0,5% (p / v) SDS pendant 3 heures. </li><li> Perfuser à l&#39;eau distillée pendant 15 minutes. </li><li> Perfuser avec 1% (p / v) de Triton X-100 dans l&#39;eau distillée pendant 30 minutes pour enlever toute les acides nucléiques liés. </li><li> Pour laver le foie décellularisé matrice (DLM), perfuser avec du PBS pendant 2 heures. </li><li> En option: réséquer tous les lobes, sauf le lobe médian. </li><li> Stocker le DLM dans un bol propre et scellé Petri trempé dans du PBS à 4 ° C jusqu&#39;au moment de servir (figure 1). </li><li> Pour stériliser le DLM: 1) Rincer le DLM avec du PBS stérile contenant 0,1% (v / v) d&#39;acide peracétique et de 4% (v / v) d&#39;éthanol et incuber pendant 3 heures à 4 o C. 2) Laver avec du PBS stérile 2 fois. 3) Laver avec du PBS stérile contenant 2% de pénicilline-streptomycine, 10ug / ml de gentamicine, et 2,5 ug / ml d&#39;amphotéricine B. Entreposer le foie décellularisé dans la même solution à 4 o C jusqu&#39;au moment de servir pour des expériences recellularization. </li></ol> 2. Recellularization du foie décellularisé Matrice <ol><li> Mettre en place un système de perfusion qui se compose d&#39;une chambre de perfusion, la pompe péristaltique et d&#39;un piège à bulles dans des conditions stériles. Remplir le système de perfusion avec 200 ml de milieu de culture, par exemple, le glucose élevé DMEM (Sigma), 10% sérum de veau foetal (Hyclone,), 100 U mL -1 pénicilline et 100 pg mL -1 streptomycine (Invitrogen). </li><li> Placer la matrice du foie décellularisé dans la chambre de perfusion et de connecter le DLM au système de perfusion à travers la canule la veine porte pendant que la pompe fonctionne à 5 mL / min pour éviter la formation de bulles d&#39;air. </li><li> Laisser la perfusion du milieu à travers le DLM pendant 30 minutes. </li><li> Isoler des hépatocytes primaires à partir d&#39;un rat adulte avec une viabilité d&#39;au moins 90%. </li><li> Arrêter l&#39;écoulement dans le système de perfusion et injecter lentement 50000000 hépatocytes (dans 1-3 ml de milieu de culture) dans le système de perfusion à travers le piège à bulles. </li><li> Démarrer le débit à 10 mL / min et recirculer la moyenne pendant 10 minutes. </li><li> Répétez les étapes 2.5 et 2.6 jusqu&#39;à ce qu&#39;un total de 200 millions de cellules sont introduites dans le DLM (figure 2) 6. </li><li> Une fois que toutes les cellules sont perfusées dans le DLM, recueillir le perfusat en quatre tubes à centrifuger de 50 ml et centrifuger à 600 rpm pendant 10 minutes. Jeter le surnageant et collecter les pastilles dans un seul tube. Déterminer le nombre de cellules et la viabilité à travers exclusion du bleu trypan pour déterminer l&#39;efficacité d&#39;ensemencement. </li></ol> 3. Culture in vitro du greffon hépatique Recellularized <ol><li> Mettre en place un système de perfusion qui se compose d&#39;une chambre de perfusion, la pompe péristaltique, oxygénateur et un piège à bulles dans des conditions stériles (figure 3). Remplir le système de perfusion avec 50 ml de milieu de culture, par exemple, E Williams (Sigma), 5% sérum de veau foetal (Hyclone,), 0,5 U mL -1 insuline (Eli Lilly), 20 ng ml -1 EGF (Invitrogen) , 14 ng ml -1 glucagon (Bedford Laboratories), 7,5 ug mL -1 d&#39;hydrocortisone (Pharmacia), 100 U mL -1 pénicilline et 100 pg mL -1 streptomycine (Invitrogen). </li><li> Placez la greffe du foie recellularized dans la chambre de perfusion et de connecter le DLM au système de perfusion à travers la canule la veine porte pendant que la pompe fonctionne à 5 mL / min pour éviter la formation de bulles d&#39;air. </li><li> Aseptiquement fermer la chambre de perfusion et fermez hermétiquement pour éviter toute fuite pendant la culture. </li><li> Transférer le système de perfusion d&#39;un incubateur est de 37 o C et a 10% de CO 2 et d&#39;augmenter le débit de perfusion à 15 mL / min. </li><li> Connectez l&#39;oxygénateur à un CO à 95% d&#39;O 2 et 5% 2 réservoir de mélange de gaz et régler le débit de gaz de 0,5 litres / min. Cela devrait atteindre une pression partielle d&#39;oxygène approximationListe des sites 400 mmHg. </li><li> La culture peut se poursuivre jusqu&#39;à 10 jours avec les changements quotidiens de milieu de culture. Le milieu de culture peuvent être prélevés quotidiennement pour la surveillance des fonctions hépatiques de la greffe comme l&#39;albumine, l&#39;urée et la sécrétion d&#39;acides biliaires totaux. A la fin de la période de la culture, la greffe du foie recellularized peuvent être échantillonnés pour l&#39;analyse moléculaire et histologique. </li></ol> 4. Les résultats représentatifs: La décellularisation complète d&#39;un foie de rat prend environ 72 heures en utilisant le protocole décrit. La matrice résultante conserve 100% du collagène fibrillaire, 50% des glycosaminoglycanes et seulement 5% de l&#39;ADN du foie natif (tableau 1) 6. La structure vasculaire de la matrice est conservée comme en témoigne la corrosion coulée et la numérisation d&#39;analyse microscopie électronique (figure 4) 6. La présence de la microarchitecture vasculaires dans le DLM facilite son repeuplement avec des cellules avec un rendement de 96% et sa perfusion subséquente de culture in vitro. La greffe du foie recellularized peuvent être cultivées jusqu&#39;à 10 jours in vitro et affiche les fonctions hépatiques bon que confirmé par l&#39;albumine, l&#39;urée et la sécrétion d&#39;acides biliaires totaux (figure 5) 6. <img src="/files/ftp_upload/2394/2394fig1.jpg" alt="Figure 1" /> Figure 1. Décellularisé matrice de foie à la fin du processus de décellularisation. (A) la totalité du foie (b) le lobe médian après résection. <img src="/files/ftp_upload/2394/2394fig2.jpg" alt="Figure 2" /> Figure 2. Représentation schématique de la recellularization du DLM. <img src="/files/ftp_upload/2394/2394fig3.jpg" alt="Figure 3" /> Figure 3. Installation du système de perfusion pour la culture in vitro de la greffe du foie recellularized. <img src="/files/ftp_upload/2394/2394fig4.jpg" alt="Figure 4" /> Figure 4. La structure microvasculaire est retenu dans la matrice du foie décellularisé. Images fonte due à la corrosion d&#39;un) un foie normal b) un foie décellularisé, portail (rouge) et veineux (bleu) vascularisation. Numérisation d&#39;images de microscopie électronique de la c DLM) un navire, d) une section avec des voies biliaires-comme les petits vaisseaux (flèches), barres d&#39;échelle (a, b) 5 mm (c, d) 20 um. <img src="/files/ftp_upload/2394/2394fig5.jpg" alt="Figure 5" /> Figure 5. Fonctions hépatiques spécifiques de la greffe du foie recellularized pendant la perfusion dans la culture in vitro. la production d&#39;urée d&#39;une sécrétion) albumine (p = 0,5249), b) (p = 0,5271) et c) la sécrétion d&#39;acides biliaires totaux (p = 0,0114). L&#39;analyse statistique de la différence entre l&#39;expérience et le contrôle a été effectué au cours de la période de 10 la culture d par le test de Friedman à 0,01 =. Les barres d&#39;erreur représentent SEM (n = 3). <table border="1"><tbody><tr><td></td><td> Frais Livera </td><td> Décellularisé foie matrice A </td><td rowspan="2"> p-valeurs </td><td rowspan="2"> % De foie frais </td></tr><tr><td></td><td> n = 4 </td><td> n = 8 </td></tr><tr><td> Collagène </td><td rowspan="2"> 0,07 ± 0,01 </td><td rowspan="2"> 0,08 ± 0,03 </td><td rowspan="2"> 0,56 </td><td rowspan="2"> 114% </td></tr><tr><td> (Mg par g de foie) </td></tr><tr><td> Les glycosaminoglycanes </td><td rowspan="2"> 73,1 ± 6,7 </td><td rowspan="2"> 34,2 ± 2,9 </td><td rowspan="2"> 0,004 </td><td rowspan="2"> 47% </td></tr><tr><td> (Mg par g de foie) </td></tr><tr><td> L&#39;ADN </td><td rowspan="2"> 14,9 ± 5,6 </td><td rowspan="2"> 0,44 ± 0,08 </td><td rowspan="2"> 3.3 10 -5 </td><td rowspan="2"> 2,9% </td></tr><tr><td> (Mg par g de foie) </td></tr></tbody></table> Tableau 1. La composition biochimique de la matrice du foie décellularisé par rapport à la foie natif. a Les valeurs sont représentées en moyenne ± SEM

<ol>
	<li>Kulig, K. M., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+tissue+engineering.">Hepatic tissue engineering.</a> Transpl Immunol. 12, 303-310 (2004).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature biotechnology. 23, 47-55 (2005).</li><li>Dahl, S. L., Koh, J., Prabhakar, V., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+native+and+engineered+arterial+scaffolds+for+transplantation.">Decellularized native and engineered arterial scaffolds for transplantation.</a> Cell Transplant. 12, 659-666 (2003).</li><li>Schechner, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+a+vascularized+human+skin+equivalent.">Engraftment of a vascularized human skin equivalent.</a> FASEB J. 17, 2250-2256 (2003).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F., F, S. <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>Uygun, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat Med. , (2010).</li></ol>

La méthode décrite ici décellularisation perfusion produit un foie entier d&#39;échafaudage qui a la même structure brute et la microarchitecture vasculaires du foie natif. L&#39;échafaud est une composition de la matrice extracellulaire semblable à du foie natif. La méthode recellularization réalise repeuplement de l&#39;échafaud avec des cellules à haut rendement et les cellules restent viables et fonctionnelles pendant la période de culture in vitro testées. Avec l&#39;ajout de cellules...

A correction was made to <a href="http://www.jove.com/Details.php?ID=2394">Decellularization and Recellularization of Whole Livers</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Nima Saeidi
instead of:
Nima Saedi.

Erratum: Decellularization and Recellularization of Whole Livers

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decellularization and recellularization of whole livers

Le foie est un organe complexe qui nécessite une perfusion constante pour la livraison de nutriments et d&#39;oxygène et l&#39;élimination des déchets afin de survivre à 1. Les efforts pour recréer ou imitent la microstructure du foie avec des motifs en utilisant l&#39;approche de l&#39;ingénierie tissulaire et des techniques de microfabrication n&#39;ont pas réussi jusqu&#39;à présent à cause de ce défi de conception. En outre, les biomatériaux synthétiques utilisés pour créer des échafaudages pour les applications de l&#39;ingénierie tissulaire du foie ont été limitées dans l&#39;induction de la régénération des tissus et la réparation due en grande partie à l&#39;absence de motifs de cellule de liaison spécifique qui induisent les fonctions cellulaires appropriées 2. Tissus décellularisé natif vaisseaux sanguins et la peau tels 3 4 d&#39;autre part ont trouvé de nombreuses applications en ingénierie tissulaire, et ont fourni une solution pratique à certains des défis. L&#39;avantage de la matrice maternelle décellularisé est qu&#39;il conserve, dans une certaine mesure, la composition originale, et la microstructure, et donc l&#39;amélioration de la fixation des cellules et la réorganisation 5. Dans ce travail, nous décrivons les méthodes pour effectuer la perfusion-décellularisation du foie, tels qu&#39;un foie intact bioscaffold qui conserve la structure des vaisseaux sanguins majeurs est obtenu. En outre, nous décrivons des méthodes pour recellularize ces biomatériaux avec un adulte hépatocytes primaires, la création d&#39;une greffe du foie qui est fonctionnel in vitro, et a l&#39;accès du navire nécessaires pour la transplantation in vivo.

Watch this Scientific Journal Video about Decellularization and Recellularization of Whole Livers at JoVE.com

Decellularization and Recellularization of Whole Livers

Décellularisation perfusion est une nouvelle technique pour produire des échafaudages foie entier qui conserve la composition de l&#39;organe de la matrice extracellulaire et la microarchitecture. Ici, la méthode de préparation d&#39;échafaudages organe entier en utilisant décellularisation perfusion et le repeuplement ultérieur avec les hépatocytes est décrite. Greffes de foie fonctionnel et transplantables peuvent être générés en utilisant cette 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 ...

decellularization-and-recellularization-of-whole-livers

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JoVE Journal

Bioengineering

21.9K vues.  Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children. Décellularisation perfusion est une nouvelle technique pour produire des échafaudages foie entier qui conserve la composition de l'organe de la matrice extracellulaire et la microarchitecture. Ici, la méthode de préparation d'échafaudages organe entier en utilisant décellularisation perfusion et le repeuplement ultérieur avec les hépatocytes est décrite. Greffes de foie fonctionnel et transplantables peuvent être générés en utilisant cette technique.

Vidéo: Decellularization and Recellularization of Whole Livers

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 ...

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Bio-ingénierie

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

Une méthodologie décellularisation pour la production d&#39;un naturel acellulaire intestinale Matrice

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

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.

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 ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Décellularisation comparable d’Explants de tissu cardiaque foetale et adulte en 3D-comme des plateformes pour les études In Vitro

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

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.

Deux méthodes de DÉCELLULARISATION de tissus végétaux pour utilisations en génie tissulaire

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

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

DÉCELLULARISATION et méthodologie de Recellularization pour les veines saphènes humaines

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

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 ...

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.

Isolement et DÉCELLULARISATION d’un pancréas entier porcin

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

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 ...