Les auteurs remercient le professeur Hendrik Tevaearai et du département de chirurgie cardiovasculaire de soutien financier pour la première partie de l&#39;étude.

REMARQUE: Lewis rats mâles et femelles, 200-220 g ont été logés dans des conditions standard de laboratoire (lumière de 12 h et le cycle sombre, l&#39;eau ad libitum et alimentaires, IVC cage). Tous les animaux ont été traités en conformité avec les recommandations de la FELASA et la loi suisse sur la protection des animaux. 1 Préparation des cellules: les cellules souches mésenchymateuses isolement de moelle osseuse <ol><li> Anesthésier les rats avec de l&#39;isoflurane à 5% de O 2 et 5 l / min dans une chambre d&#39;induction pendant 5 min. Placer le museau de l&#39;animal dans un cône d&#39;entrée connecté au système d&#39;anesthésie. Effectuer un orteil ou pincement de la queue pour confirmer la sédation. </li><li> Enlevez la peau de la jambe avec des ciseaux en coupant la peau de la cheville à la hanche. Enlever les muscles et couper l&#39;artère fémorale pour la saignée des animaux. Exposer l&#39;articulation de la hanche et disloquer la tête du fémur. Il est important de ne pas endommager la tête du fémur. Placer les os dans du PBS stérile dans un tube en plastique de 50 ml. </li><li> Retirez tous les muscles et les ligaments des os sans les casser. NOTE: Il est important que l&#39;os reste intacte pour éviter liaison de solution de rinçage ou une contamination par de l&#39;éthanol. Rincer les os pelées éthanol à 70% pendant 5 min. </li><li> Sous la hotte à flux laminaire stérile, couper les deux extrémités des os avec des ciseaux. Rincer la moelle osseuse par injection de PBS stérile de l&#39;extrémité de l&#39;os. Recueillir osseuse dans un tube de 15 ml. Centrifugeuse 7 min à 300 x g. </li><li> Eliminer le surnageant. Suspendre le culot dans 3 ml de tampon de lyse Rouge de cellules sanguines. Incuber la suspension pendant 1 min à température ambiante avant de tourner à nouveau pendant 7 minutes à 300 x g. Dans un ballon de 150 ml de culture, ajouter un milieu de culture cellulaire stérile (395 ml de milieu IMDM, 5 ml de pen / strep, 100 ml de FBS) et ensemencer les cellules. </li><li> Changer le milieu de la deuxième journée pour éliminer les cellules non-adhérentes. <ol><li> Effectuer changements de milieu tous les deux jours pendant deux à trois semaines jusqu&#39;à ce que la quantité de cellules désiré soit obtenu. </li><li> Sinon, préparer la biograft par grainesment les cellules sur la matrice solide comme nécessaire et décrit par ailleurs 3. </li></ol></li><li> Le jour de l&#39;implantation cardiaque, préparer les cellules ou récolter le biograft juste avant l&#39;application épicardique. <ol><li> Recueillir les cellules en utilisant Accutase solution de détachement cellulaire. Compter les cellules. Remplir un tube à centrifuger de 1,5 ml avec le volume de la suspension cellulaire pour obtenir une quantité calculée de la cellule souhaitée. Centrifuger pendant 7 min à 300 g et éliminer le surnageant. </li><li> Sinon, récolter le biograft de milieu de culture cellulaire, rincer dans du PBS stérile, et garder dans un milieu de culture frais sans sérum. </li></ol></li></ol> 2 Première thoracotomie et LAD ligature <ol><li> Peser le rat. Mettez le coussin chauffant à 37 ° C. Anesthésier le rat avec 5% d&#39;isoflurane et 5 l / min à 100% de O 2 dans une chambre d&#39;induction de 5 à 7 min. Effectuer un orteil ou pincement de la queue pour confirmer la sédation. Placer le rat sur le coussin chauffant. </li><li> Intuber l&#39;animal avec un 14G iv cathéter. Raccorder le cathéter d&#39;intubation à un ventilateur de rongeur programmé pour 2,5 L / min d&#39;oxygène, 2,5% d&#39;isoflurane, le volume courant de 2 ml, et une fréquence respiratoire de 90 respirations / min. </li><li> Préparer une solution de buprénorphine à 0,1 mg / kg. Injecter en sous-cutané d&#39;un tiers de la solution. Raser la partie gauche du thorax. Désinfecter avec une solution à 1% de Betadine. </li><li> Inciser la peau perpendiculairement au sternum à la quatrième espace intercostal. Séparez les 3 couches de muscle thoracique (grand pectoral, ordre croissant pectoral, et pectoral oblique externe). Ouvrir le quatrième espace intercostal (entre les nervures 4 et 5). </li><li> Utilisez un petit écarteur pour écarter les côtes et à exposer le cœur. Ouvrez le péricarde attentivement. Recherchez et ligaturer descendante antérieure gauche artère coronaire (DAL) avec un 7,0 mm suture 4 ci-dessous l&#39;atrium. </li><li> Fermez l&#39;espace intercostal avec deux points à l&#39;aide de 3,0 points de suture. Placez les deux points de suture proximale et distale du sternum. Tout d&#39;abord, tighten la suture distale. Fixer la tubulure d&#39;échappement du ventilateur pendant 2 s pour gonfler la poitrine et d&#39;éviter toute pneumothorax. Serrer la deuxième suture. Placez les couches de muscles en place (pas de sutures sont nécessaires). Fermez la peau avec une suture 5.0. </li><li> Désinfecter la suture avec une solution de Betadine 1%. Injecter le reste de la solution de buprénorphine. Éteignez le système d&#39;anesthésie. Retirer intubation cathéter. </li><li> Gardez le rat dans la cage sous une lampe chaude pendant 1-2 heures. Conserver un thermomètre dans la cage et de contrôler la distance entre la lampe et la cage chaud afin d&#39;éviter toute surchauffe. Retour le rat dans l&#39;unité IVC. Injecter par voie sous cutanée buprénorphine 0,05-0,1 mg / kg après chaque -operatively 6-12 h pendant 48 heures. </li></ol> 3. épicardique administration de la Via traitement seconde thoracotomie <ol><li> Répétez les étapes 2.1 à 2.3. </li><li> Inciser la peau perpendiculairement au sternum à la cinquième espace intercostal. Séparer la couche 3s de muscle thoracique (grand pectoral, ordre croissant pectoral et grand pectoral oblique externe). Ouvrez le cinquième espace intercostal (entre les nervures 5 et 6). </li><li> Utilisez un petit écarteur pour écarter les côtes et à exposer le cœur. Si il ya une certaine adhésion, les retirer soigneusement avec une pince fine. </li><li> Localisez la zone de l&#39;infarctus qui apparaît comme une zone pâle dessous de la ligature. REMARQUE: Si nécessaire, utiliser un morceau de 10 cm de fil de suture (7.0) inséré au sommet de mieux visualiser le ventricule gauche. Tirez doucement à la suture lâche maintenu par une pince pour exposer le cœur. </li><li> Appliquer l&#39;un des traitements suivants: <ol><li> Positionner la biograft à la surface du cœur. Utiliser la seringue pré-remplie d&#39;agent d&#39;étanchéité de fibrine préchauffée à la température ambiante pendant 10 min. Appliquez une goutte (50-100 pi) de la colle de fibrine sous la biograft aide d&#39;une aiguille 25 G Luer. Vérifiez que le biograft est parfaitement étanche. </li><li> Alternativement, appliquer le culot cellulaire recueilli avec une pipette à la surface of le cœur sur la zone pâle dessous de la ligature. Appliquer une goutte (50 à 100 pi) de la colle de fibrine sur le culot cellulaire. </li></ol></li><li> Retirez le morceau de suture à partir du sommet. </li><li> Répétez les étapes 2,6-2,8. </li></ol>

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The authors have nothing to disclose.

La ligature permanente de LAD provoque une lésion myocardique irréversible. Le premier modèle animal a été décrit en 1960 4. Depuis lors, il a été considéré comme un modèle standard et adapté à MI chronique. Sa stabilité et la reproductibilité autorisés évaluation expérimentale de thérapies pour MI 5. L&#39;amélioration des procédures suivantes la description initiale ont signalé un taux de mortalité opératoire de 35-13% 6. Comme prév...

Depuis la fin du 19 e siècle, les maladies cardiovasculaires est resté le numéro un tueur en série dans les pays industrialisés. Parmi ceux-ci, d&#39;une maladie de l&#39;artère coronaire représente l&#39;étiologie principale. Les résultats de la phase aiguë de l&#39;infarctus du myocarde (IM) et est suivi par le remodelage inadapté qui se développe progressivement vers une phase chronique et une insuffisance cardiaque sévère. Malgré les progrès technologiques et thérapeutiques significatifs récents, la morbidité et la mortalité dues à la progression de l&#39;insuffisance cardiaque est encore en croissance 1. Dans ce contexte, la thérapie cellulaire a acquis un intérêt croissant en tant que nouvelle option thérapeutique pour stopper la progression de la maladie vers l&#39;insuffisance cardiaque et à stimuler la capacité de régénération a récemment identifié du myocarde. Des études expérimentales et cliniques ont fourni des preuves convaincantes des effets bénéfiques obtenus après transplantation cardiaque de divers types de cellules. Les principaux résultats figurent l&#39;amélioration de cardila fonction contractile alternatif, une diminution de remodelage ventriculaire gauche, diminution de la taille de l&#39;infarctus, et a augmenté la densité vasculaire dans la zone infarcie. Toutefois, le nombre de cellules à faible rétention après l&#39;injection des cellules est resté un inconvénient important. Association de cellules avec une biomatrice pour améliorer la prestation de la cellule 2 ont récemment favorisé chercheur et intérêts cliniques. La ligature de la descendante antérieure gauche de l&#39;artère coronaire (DAL) est une méthode de référence pour MI en petit modèle animal qui se traduit par infarctus transmural et une cicatrice mature. La thérapie cellulaire appliqué dans la phase chronique de la MI nécessite une deuxième intervention chirurgicale. Une sternotomie médiane est généralement réalisée pour permettre l&#39;injection intramyocardique de l&#39;implantation des cellules ou de biografts épicardique. Ces procédures chirurgicales invasives augmentent le taux de mortalité post-opératoire temps de récupération, la douleur et le risque d&#39;infection. L&#39;approche mini-invasive présentée ici non seulement empêche une telle partialité, mais fournit également des oaccessibilité ptimal du cœur pour l&#39;application du traitement. MI et l&#39;implantation épicardique de cellules associées à une biomatrice de type gel sont effectuées sur un coeur battant par thoracotomie intercostale gauche.

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			<td style="width:239px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:216px;">POLYSORB 3-0 suture</td>
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			<td></td>
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			<td height="21" style="height:21px;width:216px;">SURGIPRO II 7-0 Suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">VP904X</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">CATHETER INSYTE 14G</td>
			<td style="width:104px;">BD</td>
			<td style="width:119px;">381267</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">557058</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
			<td style="width:104px;">FST Germany</td>
			<td style="width:119px;">11274-20</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibrin sealant</td>
			<td style="width:104px;">BAXTER</td>
			<td style="width:119px;">1501441</td>
			<td>TISSEEL Glue, Frozen -20&#176;C</td>
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			<td height="42" style="height:42px;width:216px;">CASTROVIEJO EYE SPECULA</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">72-8925</td>
			<td>use as retractor for the ribs</td>
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			<td height="21" style="height:21px;">IMDM Glutamax</td>
			<td>Gibco</td>
			<td>31980</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>PAA Clone</td>
			<td>&#160;&#160;&#160;&#160;&#160; A15-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">bone scissors</td>
			<td style="width:104px;">Fine Science Tols</td>
			<td style="width:119px;">16044-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red Blood Cell Lysis Solution</td>
			<td>Gentra Systems</td>
			<td>D-50k1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
			<td style="width:104px;">Stem cell technology</td>
			<td style="width:119px;">7920</td>
			<td></td>
		</tr>
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cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix

La thérapie cellulaire cardiaque a suscité un intérêt et l&#39;implantation de biomatériaux associés à des cellules de plus en plus est devenu un enjeu majeur pour optimiser la prestation des cellules myocardiques. modèle de rongeur de l&#39;infarctus du myocarde (IM) constitué de gauche artère interventriculaire antérieure (AIA) ligature a généralement été réalisée via une thoracotomie; une deuxième opération à cœur ouvert via une sternotomie est traditionnellement réalisée à une application épicardique du traitement. Depuis la description de modèle de ligature LAD, le taux de mortalité post-opératoire a chuté de 35 à 13%, mais la deuxième chirurgie est restée critique. Afin d&#39;améliorer la récupération post-opératoire et de réduire la douleur et l&#39;infection, les procédures chirurgicales minimalement invasives sont présentés. Deux thoracotomie ont été réalisées, une première pour la ligature LAD et le second pour le traitement administration épicardique. Biografts constitués de cellules associées à des matrices solides ou de type gel ont été appliqués sur l&#39;ar infarciech. CONT ligature a entraîné la perte de la fonction cardiaque telle que confirmée par échocardiographie réalisée au bout de 2 et 6 semaines. Goldner coloration au trichrome effectué sur des sections de coeur confirmé la formation de cicatrices transmurale. Des premier et second chirurgies entraîné moins de 10% de la mortalité post-opératoire.

Tous les animaux ont récupéré dans 1 heure après thoracotomie. La cicatrisation des plaies a été rapide. Aucune infection ou un oedème a été observé. La thoracotomie gauche double a permis un accès optimal à cœur (Figure 1). La douleur et la mortalité post-opératoire était faible. L&#39;animal a récupéré rapidement de la chirurgie et a pris du poids (figure 2). Kaplan Meir pour cent de survie était de 96% pour la première thoracotomie. Q...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

L&#39;implantation d&#39;un biograft pour traiter l&#39;infarctus du myocarde induite par la ligature LAD dans un modèle de rongeur est classiquement requise des deux opérations à cœur ouvert. Afin de réduire la mortalité et de fournir des conditions optimales pour la fixation de biomatrices gélatineuses solides et associés à des cellules, des procédures minimalement invasives ont été développées.

Thérapie à base de cellules pour l&#39;insuffisance cardiaque chez le rat: Double thoracotomie pour infarctus du myocarde et épicardique implantation de cellules et Biomatrice

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

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Research

JoVE Journal

Bioengineering

11.8K vues.  University of Fribourg.  L'implantation d'un biograft pour traiter l'infarctus du myocarde induite par la ligature LAD dans un modèle de rongeur est classiquement requise des deux opérations à cœur ouvert. Afin de réduire la mortalité et de fournir des conditions optimales pour la fixation de biomatrices gélatineuses solides et associés à des cellules, des procédures minimalement invasives ont été développées. 

Vidéo: Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Imagerie par bioluminescence pour l&#39;évaluation des réponses immunitaires après l&#39;implantation du tissu cardiaque d&#39;ingénierie (ISE)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

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Bio-ingénierie

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplantation de progéniteurs myoangioblastes dérivés de cellules souches pluripotentes induites dans des modèles murins de régénération musculaire

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

Analyse de la membrane tubulaire Networks cardiaque myocytes de oreillettes et les ventricules

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Ensemencement et Implantation d’un greffon biosynthétique de trachéale tissulaire dans un modèle murin

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Évaluation de la prolifération des kératinocytes sur deux et trois dimensions des substrats de collagène de Type I

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

Microbulles magnétiques, acoustiques et optiques triple-réactives pour l’hyperthermie magnétique et la thérapie du cancer combinée pothototherme

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Préparation de tissus cardiaques en forme de maille dérivés de cellules iPS humaines pour la réparation myocardique in vivo

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Transplantation d’un patch bioimprimé 3D dans un modèle murine d’infarctus du myocarde

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Quantification de la fonction ventriculaire gauche du cœur de souris, de la tension myocardique et des forces hémodynamiques par imagerie par résonance magnétique cardiovasculaire

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Évaluation de la thérapie de modulation de la contractilité cardiaque dans des cardiomyocytes 2D dérivés de cellules souches humaines

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...