Nous remercions Maegan B. Colehour de l&#39;aide pour le développement bioréacteur. Ces études ont été financées par l&#39;Université de Yale Département d&#39;anesthésie et par NIH 098220 HL (à LEN). THP a été soutenu par le NIH T32 GM007171.

<ol>
	<li>American Lung Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lung Disease Data. , (2008).</li><li>Petersen, T. H., Calle, E. A., Colehour, M. B., 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=Bioreactor+for+the+Long+Term+Culture+of+Lung+Tissue.">Bioreactor for the Long Term Culture of Lung Tissue.</a> Cell Transplant. , (2010).</li><li>Bilek, A. M., Dee, K. C., Gaver, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+surface+tension+induced+epithelial+cell+damage+in+a+model+of+pulmonary+airway+reopening.">Mechanisms of surface tension induced epithelial cell damage in a model of pulmonary airway reopening.</a> J Appl Physiol. 94, 770-783 (2003).</li><li>Petersen, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-Engineerined+Lungs+for+in+Vivo+Implantation.">Tissue-Engineerined Lungs for in Vivo Implantation.</a> Science. 329, 538-5341 (2010).</li><li>Ott, H. C. <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, 927-9233 (2010).</li><li>Cortiella, J. <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 (2010).</li><li>Sugihara, H., Toda, S., Miyabara, S., Fujiuyama, C., Yonemitsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+alveolus-like+structure+from+alveolar+type+II+epithelial+cells+in+three-dimensional+collagen+gel+matrix+culture.">Reconstruction of alveolus-like structure from alveolar type II epithelial cells in three-dimensional collagen gel matrix culture.</a> Am J Pathol. 142, 783-7892 (1993).</li><li>Choe, M. M., Sporn, P. H., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+remodeling+by+dynamic+strain+in+a+three-dimensional+tissue-engineered+human+airway+wall+model.">Extracellular matrix remodeling by dynamic strain in a three-dimensional tissue-engineered human airway wall model.</a> American Journal Respiratory Cell Molecular Biology. 35, 306-306 (2006).</li><li>Cortiella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-enginered+lung:+an+in+vivo+and+in+vitro+comparison+of+polyglycolic+acid+and+pluronic+F-127+hydrogel/somatic+lung+progenitor+cell+constructs+to+support+tissue+growth.">Tissue-enginered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth.</a> Tissue Engineering. 12, 1213-1213 (2006).</li><li>Price, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Decellularized+Lung+Bioreactor+System+for+Bioengineering+the+Lung:+The+Matrix+Reloaded.">Development of a Decellularized Lung Bioreactor System for Bioengineering the Lung: The Matrix Reloaded.</a> Tissue Eng Part A. , (2010).</li><li>Ott, H. C. <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. 14, 213-221 (2008).</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. 16, 814-820 (2010).</li><li>Shannon, J. M., Mason, R. J., Jennings, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differentiation+of+alveolar+type+II+epithelial+cells+in+vitro:+effects+of+cell+shape,+cell-matrix+interactions+and+cell-cell+interactions.">Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions.</a> Biochim. Biophys. Acta. 931, 143-156 (1987).</li><li>Cortiella, J. <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. , (2010).</li></ol>

LEN détient des titres dans Humacyte, une société de médecine régénérative. Humacyte ne pas financer ces études, et n&#39;a pas d&#39;incidence sur la conception, l&#39;interprétation, ou la déclaration de l&#39;un des expériences ou des méthodes décrites. Les auteurs (LEN, THP, EAC) et l&#39;Université de Yale ont déposé une demande de brevet relatifs à l&#39;ingénierie des tissus des poumons.

Les aspects les plus critiques du système présentés ici incluent maintien de la stérilité, et une surveillance étroite de pressions appliquées sur le lit vasculaire à travers le processus de préparation et l&#39;ensemencement de l&#39;échafaud et la culture des poumons repeuplé. La stérilité est mieux maintenu par autoclavage tous les matériaux avant leur utilisation, ainsi que par le montage du poumon dans un système fermé, peu après explant et évitant violation ultérieure de cette barrière. Une fois la matrice décellularisé a été soigneusement rincés et transférés dans un bioréacteur stérile pour la culture, le bouchon de silicone et autres phoques et les connexions ne devraient pas être dérangé ou enlevé. Pour maintenir la pression en échec, le débit conduit par gravité est préférable lorsque cela est possible. Quand une pompe est nécessaire pour la recirculation du liquide, en commençant après le rinçage avec du PBS et continue au cours de la culture, nous vous recommandons de mesurer directement la pression juste avant que le fluide pénètre dans l&#39;artère pulmonaire avec un capteur de pression. L&#39;ampleur de la pression appliquée doit pas dépasser 15 mm Hg. Poumons Recellularized peuvent être cultivées pour différentes périodes, allant généralement de 4 jours à 3 semaines au maximum. Perfusion vasculaire est généralement réalisé à 1-3 ml / min pendant la culture endothéliales, tandis que la ventilation est généralement appliquée à un taux de 1 respiration / min pendant la culture épithéliales. Pendant les périodes de culture associée, la ventilation et la perfusion simultanée est approprié. La ventilation peut être effectuée soit avec un milieu liquide ou par air. Au cours de ces dernières décennies, plusieurs groupes ont réalisé d&#39;importants travaux d&#39;ingénierie tissulaire démontrant la faisabilité de différencier l&#39;épithélium pulmonaire in vitro et de reproduire plusieurs aspects du poumon microanatomie 7,8,9, 10. Cependant, jusqu&#39;à récemment, aucune de ces tentatives 4,5 pour concevoir les tissus pulmonaires avaient abouti à un organe implantables qui a été en mesure de maintenir la séparation entre le sang et les compartiments des voies aériennes et qui pourraient participer à l&#39;échange gazeux. Par conséquent, si les méthodes décrites ne sont qu&#39;une première étape vers l&#39;objectif à long terme de générer des tissus pulmonaires fonctionnels, ce travail est une étape encourageante vers la possibilité d&#39;augmenter la quantité de tissu pulmonaire disponibles pour la transplantation. Par ailleurs, ce travail élabore travail effectué par Ott et al. Uygun et ses collègues et de 11,12, ce qui démontre l&#39;efficacité d&#39;une matrice extracellulaire décellularisé comme un échafaudage pour l&#39;ingénierie tissulaire complexes en trois dimensions des structures et de soutenir la croissance et la survie de différents types de cellules . Ce travail est également importante pour sa contribution à l&#39;arsenal des cellules respiratoires et biologistes moléculaires. En fournissant un unique environnement tridimensionnel qui peut également fournir des stimuli mécaniques, et qui ne partage pas le risque d&#39;accompagnateur rapidement dé-différenciation que l&#39;on pourrait rencontrer lors de l&#39;épithélium de rongeurs en culture en laboratoire avec des méthodes plus traditionnelles 13, les scientifiques pourraient utiliser notre système pour gagner un nouvel éclairage sur les interactions cellule-cellule et cellule-matrice qui jouent un rôle dans la différenciation cellulaire et la fonction. Cette connaissance peut être particulièrement puissant si utilisé comme levier pour orienter le destin des différentes populations de cellules souches, en tant que groupe Cortiella a démontré dans les études initiales 14.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> L&#39;euthanasie </td><td></td><td></td><td></td></tr><tr><td> Héparine </td><td> Sigma-Aldrich </td><td> H4784 </td><td></td></tr><tr><td> Nitroprussiate de sodium </td><td> Fluka </td><td> 71778 </td><td></td></tr><tr><td> Solution de l&#39;euthanasie Euthasol </td><td> Virbac AH </td><td> 710101 </td><td> 390 mg / ml, solution de pentobarbital de sodium stocks doivent être dilués de façon appropriée pour l&#39;administration IP </td></tr><tr><td colspan="4"></td></tr><tr><td> Solution Decell </td><td></td><td></td><td></td></tr><tr><td> CHAPS </td><td> Sigma-Aldrich </td><td> C3023 </td><td></td></tr><tr><td> NaCl </td><td> Américaine bioanalytiques </td><td> AB01915 </td><td></td></tr><tr><td> EDTA </td><td> Sigma </td><td> E5134 </td><td></td></tr><tr><td> NaOH </td><td> JT Baker </td><td> 3722-01 </td><td></td></tr><tr><td> PBS 1X </td><td> Gibco </td><td> 14190 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td> Composants bioréacteur </td><td></td><td></td><td></td></tr><tr><td> 480 ml pot </td><td> Cole Parmer </td><td> EW-3460560 </td><td></td></tr><tr><td> Bouchon en silicone, taille 14 </td><td> Cole Parmer </td><td> EW-06298-26 </td><td></td></tr><tr><td> Y connecteurs </td><td> Cole Parmer </td><td> ED-30614-08 </td><td> Utilisé pour les canules artérielles et trachéaux </td></tr><tr><td> Tube silicone </td><td> Masterflex </td><td> 96420-14; 16 </td><td> L / S 14, 16 </td></tr><tr><td> Transducteurs de pression </td><td> Edwards Lifesciences </td><td> PX-212 </td><td></td></tr><tr><td> Clapet </td><td> Cole Parmer </td><td> EW-98553-20 </td><td> Un clapet anti-retour </td></tr><tr><td> À 4 voies robinets </td><td> Edwards Lifesciences </td><td> 594WSC </td><td></td></tr><tr><td> Filtre seringue </td><td> Cole Parmer </td><td> 2915-08 </td><td> PTFE, 0,2 um </td></tr><tr><td colspan="4"></td></tr><tr><td> Decell et apparati rinçage (ajouts à bioréacteur) </td><td></td><td></td><td></td></tr><tr><td> 500 et 1000 ml bouteilles en verre </td><td> Corning </td><td> 1395-500;-1L </td><td> Utilisé pour decell et rinçage par gravité </td></tr><tr><td colspan="4"></td></tr><tr><td> Traitement Benzonase </td><td></td><td></td><td></td></tr><tr><td> Pénicilline / streptomycine </td><td> Gibco </td><td> 15140122 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.03 </td><td></td></tr><tr><td> Tris-HCl </td><td> Américaine bioanalytiques </td><td> AB14043 </td><td> 1M, pH 8,0 </td></tr><tr><td> MgCl 2 </td><td> JT Baker </td><td> 2444-01 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A9647 </td><td></td></tr><tr><td> Benzonase Nuclease </td><td> Sigma </td><td> E1014 </td><td> Endonucléase utilisée pour enlever vestige d&#39;ADN de la matrice </td></tr></tbody></table>

procedure for lung engineering

Le tissu pulmonaire, y compris le cancer du poumon et les maladies pulmonaires chroniques telles que la bronchopneumopathie chronique obstructive, cumulativement représentent environ 280 000 décès par an; maladie pulmonaire obstructive chronique est actuellement la quatrième cause principale de décès aux États-Unis 1. Contribuer à cette mortalité est le fait que les poumons ne sont généralement pas au-delà de réparer ou de régénérer le microscopique, au niveau cellulaire. Par conséquent, le tissu pulmonaire qui est endommagé par une dégénérescence ou une infection, ou du tissu pulmonaire qui est une résection chirurgicale n&#39;est pas fonctionnellement remplacé in vivo. Pour déterminer si les tissus pulmonaires peuvent être générés in vitro, nous avons traité les poumons de rats adultes en utilisant une procédure qui supprime les composants cellulaires pour produire une matrice extracellulaire pulmonaire acellulaire échafaud. Cet échafaudage conserve les structures hiérarchiques de branchement des voies respiratoires et le système vasculaire, ainsi que d&#39;une membrane basale largement intact, ce qui comprend le collagène IV, laminine, fibronectine et. L&#39;échafaud est monté dans un bioréacteur conçu pour imiter les aspects critiques de la physiologie des poumons, telles que la ventilation à pression négative et de perfusion vasculaire pulsatile. En cultivant l&#39;épithélium pulmonaire et l&#39;endothélium vasculaire au sein de l&#39;échafaudage monté bioréacteur, nous sommes en mesure de générer des tissus pulmonaires qui est phénotypiquement comparables au tissu pulmonaire native et qui est capable de participer à un échange de gaz pour des intervalles de temps courts (de 45 à 120 minutes). Ces résultats sont encourageants et suggèrent que le repeuplement du poumon matrice est une stratégie viable pour la régénération du poumon. Cette possibilité constitue une occasion non seulement de travailler à augmenter l&#39;offre de tissu pulmonaire pour une transplantation, mais aussi à l&#39;étude des cellules des voies respiratoires et la biologie moléculaire in vitro pour des périodes plus longues et dans un microenvironnement plus précis que ce qui a été possible auparavant.

Watch this Scientific Journal Video about Procedure for Lung Engineering at JoVE.com

Procedure for Lung Engineering

Nous avons développé une matrice extracellulaire et des poumons décellularisé bioréacteur roman biomimétiques qui peuvent être utilisés pour générer des tissus pulmonaires fonctionnelles. Par ensemencement des cellules dans la matrice et la culture dans le bioréacteur, nous générons des tissus qui démontre les échanges gazeux efficaces lorsqu&#39;elles sont transplantées in vivo pour de courtes périodes de temps.

Procédure pour l&#39;ingénierie du poumon

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Research

JoVE Journal

Bioengineering

46.5K vues.  Yale University. Nous avons développé une matrice extracellulaire et des poumons décellularisé bioréacteur roman biomimétiques qui peuvent être utilisés pour générer des tissus pulmonaires fonctionnelles. Par ensemencement des cellules dans la matrice et la culture dans le bioréacteur, nous générons des tissus qui démontre les échanges gazeux efficaces lorsqu'elles sont transplantées in vivo pour de courtes périodes de temps.

Vidéo: Procedure for Lung Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS élastomère échafaudages en génie tissulaire artérielle

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Recherche

Bio-ingénierie

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Traitement post-production des fibres électrofilées de génie tissulaire

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capillaire de travail lithographie pour l&#39;ingénierie tissulaire cardiaque

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingénierie 3D cellularisé collagène Gels pour la régénération tissulaire vasculaire

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Synthèse de nanofibres à base de kératine de génie biomédical

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Microfluidique Bioprinting génie vascularisé tissus et organoïdes

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

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 ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Cathéter de lumière à tir latéral intra-cardiaque pour la surveillance du métabolisme cellulaire à l'aide de la spectroscopie d'absorption transmurale des cœurs de mammifères perfusés

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

Tissus pulmonaires modifiés préparés à partir de tranches pulmonaires décellularisées

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Etude comparative des techniques de décellularisation des hydrogels ECM pulmonaires bovins

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

Pleins feux sur l’auteur : Optimisation de la décellularisation pulmonaire bovine pour les hydrogels organotypiques

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

Développement et caractérisation d’hydrogels à matrice extracellulaire pulmonaire décellularisée

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...