Préparation pour la chirurgie de la partie I. <ol><li> La veille de la chirurgie chez le rat, assurez-vous que toutes les solutions sont faites, mais n&#39;ajoutez pas encore les enzymes. Tampons A, B et le tampon de lavage devraient tous être filtrée pour la stérilité avant le stockage. Les tampons devraient tous être conservés à 4 ° C. </li><li> Plaques de 6 puits de culture de tissus doivent être plaqués avec 10-20 mg / ml de laminine dans 2 mL 1XSFM la veille aussi, et doivent être conservés à 37 ° C dans un incubateur à CO 2. </li><li> Le jour de la chirurgie, la préparation des instruments chirurgicaux en stérilisant la pince, ciseaux, pinces et de l&#39;éthanol à 70% et laissez-les sécher sur du papier absorbant. </li><li> Une seringue de 1 ml remplie de 0,5 ml solution d&#39;héparine (90 U / ml) est prêt. </li><li> Maintenant, il est de bonne pratique pour laver l&#39;appareil de perfusion en parcourant 70% d&#39;éthanol, en utilisant la pompe péristaltique dans le sens inverse. Une fois terminé le rinçage avec de l&#39;éthanol, rincer l&#39;appareil avec de l&#39;eau bidistillée, et enfin rincer avec le tampon A. Le débit à travers est recueillie dans un bêcher placé dans un bain à 37 ° C l&#39;eau. </li><li> Pour préparer l&#39;appareil de perfusion nettoyer, remplir le tube droit avec une seringue de 40 ml de tampon A et le tube gauche avec une seringue de 40 ml de tampon B. </li><li> Premier de la tubulure de perfusion à l&#39;extrémité du cathéter, en permettant aux flux de tampon A travers l&#39;appareil. Recharge de la mémoire tampon d&#39;une seringue pour le niveau 40 ml. Assurez-vous qu&#39;il n&#39;ya pas de bulles d&#39;air piégées dans la tubulure après cette étape. </li><li> Maintenant, fermez le robinet vanne de tampon A seringue et répétez le processus avec tampon B de telle sorte que la tubulure de perfusion est maintenant amorcée avec le tampon B. </li><li> Enfin, le dispositif de perfusion est laissé avec le tampon B robinet ouvert la vanne, mais avec l&#39;écoulement a cessé d&#39;utiliser le régulateur sur la ligne IV. </li><li> Jeter le tampon accréditives dans le bécher de collecte. </li><li> La dernière chose à faire avant la chirurgie est d&#39;ajouter les enzymes nécessaires à votre solution de réserve, puis filtrer la solution enzymatique tout comme les autres solutions ont été filtrées de la veille. Une fois filtrée, cette solution peut être laissé à température ambiante. </li></ol> Partie II. La dissection du cœur de rat <ol><li> Après le protocole standard euthanasier le rat avec de l&#39;isoflurane dans une chambre à gaz. </li><li> Stériliser la zone d&#39;incision sur le thorax avec de l&#39;éthanol à 70%. </li><li> Ouvrez la poitrine à l&#39;aide de ciseaux et d&#39;exposer le cœur. </li><li> Injecter ventricule gauche, avec la solution de 0,5 ml d&#39;héparine (90 U / ml). </li><li> Retirer le cœur avec grande portion de l&#39;aorte intacte, le placer dans la glace froide B. Tampon </li></ol> Partie III. L&#39;isolement des myocytes <ol><li> Un cœur de rat typique devrait donner une fraction élevée de la tige en forme de myocytes (par opposition aux cellules mortes arrondi) si la procédure suivante est correctement respectée. </li><li> Pour commencer, pince aorte pour le cathéter. L&#39;extrémité du cathéter ne doit pas être poussée trop loin dans le coeur afin d&#39;assurer une bonne perfusion du cœur par l&#39;artère coronaire. </li><li> Une fois serré, la cravate de l&#39;aorte au cathéter avec une suture. </li><li> Ensuite, ouvrez le régulateur ligne IV pour permettre à un taux d&#39;égouttement rapide (~ 60 gouttes / min) et permettre tampon B pour laver le cœur. </li><li> Pendant Tampon B laver, utiliser les petits ciseaux et des pinces pour enlever les oreillettes et les tissus adipeux ou des poumons s&#39;accrochant au cœur. </li><li> Après tampon B est fini, passer le flux au tampon A. </li><li> Alors que le tampon A est autorisé à circuler pendant 5 minutes, plusieurs petites tâches doivent être réalisées très rapidement. </li><li> Tout d&#39;abord, chauffer la solution enzymatique dans un bain d&#39;eau à 37 ° C. </li><li> Enfin, lorsque le tampon A est épuisée de la seringue (mais toujours présents dans le tube), une charge de 50 ml de la solution enzymatique dans la seringue A de l&#39;appareil de perfusion </li><li> Il est maintenant nécessaire de jeter tous les flux à travers le gobelet dans le bain d&#39;eau (par pipetage), jusqu&#39;à ce que le coeur perfuse Solution enzymatique. </li><li> Dès que la solution enzymatique perfuse le cœur, d&#39;activer la pompe péristaltique qui est mis en place pour transférer la solution enzymatique du bécher collecte pour reconstituer la seringue A. </li><li> Laisser la solution enzymatique de circuler à travers le coeur pendant 10 min. Comme le cœur digère, elle commence à regarder de ballonnement. </li><li> Ajouter 37,5 ul de 0,1 M CaCl 2 à la solution enzymatique dans la seringue A pour donner une concentration effective de 0,1 mM de Ca 2 +. </li><li> Après 10 minutes d&#39;augmenter la concentration de calcium à 0,2 mM en ajoutant un supplément de 50 ul de 0,1 M CaCl 2 à la seringue A et laissez-la perfusion procéder pour encore 10 minutes. </li><li> Coupez les ventricules et les transférer à un petit bécher stérile contenant 20 ml de solution enzymatique. </li><li> Dans le bécher augmenter la concentration de calcium à 0,4 mm par ajout de 40 uL plus de 0,1 M CaCl 2. </li><li> Doucement mâche le cœur en 10 pièces ou plus avec une paire de petits ciseaux stériles. </li><li> Incuber le bécher pendant 5 minutes à 37 ° C avec doux balancement. </li><li> Ajouter 40 ml de 0,1 M CaCl 2 àdonner à la concentration effective de 0,6 mM de Ca 2 + et doucement triturer les morceaux du cœur avec une pipette de transfert en plastique 3-5 fois. </li><li> Après une incubation de 5 minutes supplémentaires à 37 ° C, ajouter 40 ml de 0,1 M CaCl 2 et doucement triturer comme avant. </li><li> Séparée digérés myocytes unique à partir de non-digérée en utilisant des tissus conjonctifs filtration à travers un grillage stériles 500 um. </li><li> Laisser les cellules sédimenter dans un tube de 50 ml pendant 10 minutes à température ambiante. </li><li> Rejeter le surnageant avec une pipette de transfert. </li><li> Resuspendre délicatement les cellules dans le tampon de lavage n ° 1 et permettre aux cellules de se contenter de 10-20 min à température ambiante. </li><li> Alors que les cellules sont de décantation, de prendre une petite aliquote, et utiliser ce temps comme une occasion d&#39;évaluer la qualité et la viabilité des cellules en suspension sous un microscope inversé. Une bonne préparation aura une proportion élevée (&gt; 80%) de la tige comme des cellules avec des stries nettes. </li><li> Une fois que les cellules se sont installés, jeter le surnageant des cellules et doucement remettre en suspension dans le tampon de lavage n ° 2. Laissez-les cellules de régler à la température ambiante, ce qui devrait à nouveau prendre 10-20 minutes, et jeter le surnageant. </li><li> Répétez le processus de lavage une fois de plus avec le tampon de lavage n ° 3, et les cellules seront prêtes pour la culture. </li></ol> Partie IV. Culture cellulaire des myocytes <ol><li> Resuspendre délicatement les cellules dans 5% des médias du sérum. Avant de transférer les cellules à plaques de culture tissulaire, se laver les plaques avec 1x GDF. Transférer les cellules à une densité désirée et incuber dans un CO 2 de culture de tissu incubateur pour 3-5 heures. </li><li> Commutateur les médias à 1x GDF. </li><li> Les cellules peuvent être cultivées pour un maximum de quatre jours et utilisés au besoin pour les expériences. </li></ol> Partie V. Représentant Résultats / Résultats Il devrait y avoir plus de 70% des myocytes cardiaques vivent sous microscope inversé lorsque le protocole est fait correctement. <img src="/files/ftp_upload/1308/fig1.jpg" alt="Figure 1" /> Tableau 1: Recette de solution pour 1 L 10X Ca 2 + sans tampon KH: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> de masse </td><td> Concentration finale </td></tr><tr><td> NaCl </td><td> 68,96 g </td><td> 1180 mm </td></tr><tr><td> KCl </td><td> 3,58 g </td><td> 48 mM </td></tr><tr><td> HEPES </td><td> 59,58 g </td><td> 250 mM </td></tr><tr><td> K 2 HPO 4 </td><td> 2,85 g </td><td> 12,5 mM </td></tr><tr><td> MgSO 4 </td><td> 3,08 g </td><td> 12,5 mM </td></tr></tbody></table> Tableau 2: Recette de solution pour une mémoire tampon L A: Ajouter 1,98 g de glucose à 100 mL de tampon KH 10X et porter à un volume final de 1 L. Ajuster osmolarité à l&#39;état de la physiologie proche (~ 300) avec du glucose. Amener à pH 7,4 avec NaOH. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> - </td><td> Masse </td><td> Concentration finale </td></tr><tr><td> Tampon 10X KH </td><td> 100 ml </td><td> 100 ml </td></tr><tr><td> Le glucose </td><td> 1,98 g </td><td> 11 mM </td></tr><tr><td> Porter à 1 L </td><td></td><td></td></tr><tr><td> Amener à pH 7,4 avec NaOH </td><td></td><td></td></tr><tr><td colspan="3"> Ajustez osmolarité à l&#39;état physiologique proche (~ 300) avec du glucose. </td></tr></tbody></table> Tableau 3: Recette Solution pour tampons enzymatiques <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> de masse </td><td> Concentration finale </td></tr><tr><td> Collagénase de type II </td><td> 25 mg </td><td> 0,05% (p / v) </td></tr><tr><td> Protéase XIV </td><td> 10 mg </td><td> 0,02% (p / v) </td></tr><tr><td> BDM </td><td> 0,025 g </td><td> 5 mM </td></tr><tr><td> Carnitine </td><td> 0,020 g </td><td> 2 mM </td></tr><tr><td> Taurine </td><td> 0,031 g </td><td> 5 mM </td></tr><tr><td> L&#39;acide glutamique </td><td> 0,020 g </td><td> 2 mM </td></tr><tr><td> 0,1 M CaCl 2 </td><td> 12,5 uL </td><td> 25 uM </td></tr><tr><td> Tampon A </td><td></td><td> Remplissez à 50 ml </td></tr></tbody></table> Tableau 4: Recette Solution pour 25X BDM / taurine / BSA (B / T / B) <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> de masse </td><td> Concentration finale </td></tr><tr><td> BDM </td><td> 0,076 g </td><td> 125 mM </td></tr><tr><td> Taurine </td><td> 0,094 g </td><td> 125 mM </td></tr><tr><td> BSA </td><td></td><td> 0,1% (p / v) </td></tr><tr><td> Tampon A </td><td></td><td> Remplissez à 6 mL </td></tr></tbody></table> Tableau 5: Recette Solution B Tampon et tampons de lavage <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tampon: </td><td> B </td><td> # 1 </td><td> # 2 </td><td> # 3 </td></tr><tr><td> Ajouter: </td><td></td><td></td><td></td><td></td></tr><tr><td> 0,5 M CaCl 2 </td><td> 0,4 ml </td><td> 0,1 ml </td><td> 0,125 ml </td><td> 0,15 mL </td></tr><tr><td> La concentration finale de CaCl 2 </td><td> 1 mM </td><td> 1 mM </td><td> 1,25 mM </td><td> 1,5 mM </td></tr><tr><td> 25X B / T / B </td><td> --- </td><td> 2 ml </td><td> 2 ml </td><td> 2 ml </td></tr><tr><td> Un volume tampon pour la finale </td><td> 200 ml </td><td> 50 ml </td><td> 50 ml </td><td> 50 ml </td></tr></tbody></table> Tableau 6: Recette Solution for Media Culture <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 2X milieu sans sérum (SFM) </td><td></td><td></td></tr><tr><td rowspan="2"> </td><td rowspan="2"></td><td> Concentration finale </td></tr><tr><td> (Après dilution) </td></tr><tr><td> Carnitine </td><td> 0,099 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Taurine </td><td> 0,063 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Créatine </td><td> 0,075 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Antibiotique / antimycotique </td><td> 0,5 ml </td><td> 1% (0,5%) (v / v) </td></tr><tr><td> Media 199 </td><td> 45 ml </td><td> Remplissez à 50 mL </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X GDF </td><td></td><td></td></tr><tr><td></td><td></td><td> Concentration finale </td></tr><tr><td> 2X GDF </td><td> 25 ml </td><td> 1X </td></tr><tr><td> Media 199 </td><td></td><td> Remplissez à 50 mL </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X 5% de sérum médias </td><td></td><td></td></tr><tr><td></td><td></td><td> Concentration finale </td></tr><tr><td> 2X GDF </td><td> 25 ml </td><td> 1X </td></tr><tr><td> FBS </td><td> 2,5 ml </td><td> 5% (v / v) </td></tr><tr><td> Media 199 </td><td></td><td> Remplissez à 50 mL </td></tr></tbody></table>

<ol>
	<li>Miriyala, J., Nguyen, T., Yue, D. T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CaVbeta+subunits,+and+lack+of+functional+reserve,+in+protein+kinase+A+modulation+of+cardiac+CaV1.2+channels.">Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.</a> Circ Res. 102 (7), e54-e64 (2008).</li><li>SX, T. a. k. a. h. a. s. h. i., Mittman, S., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+modulatory+effects+of+five+human+auxiliary+beta2+subunit+splice+variants+on+L-type+calcium+channel+gating.">Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.</a> Biophys. , 84-845 (2003).</li></ol>

Une étape essentielle est la vitesse avec laquelle le cœur isolé est accroché sur le système de perfusion. La longueur de la période de digestion enzymatique peut être un peu différent pour chaque rat. L&#39;ajustement dépend de la manière douce le cœur devient après la période régulière de la digestion. La reprise lente de Ca 2 + après digestion enzymatique est essentielle pour l&#39;obtention de Ca 2 +-tolérants aux cellules saines. Pour cochon de Gui...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9666</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63068</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7902</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4784</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>Protease XIV</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5147</td>
<td>&nbsp;</td>
<tr>
<td>2,3-Butanedione Monoxime (BDM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0753</td>
<td>&nbsp;</td>
<tr>
<td>Carnitine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C9500</td>
<td>&nbsp;</td>
<tr>
<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T0625</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3294</td>
<td>&nbsp;</td>
<tr>
<td>Creatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3630</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-009-CI</td>
<td>&nbsp;</td>
<tr>
<td>Media 199</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11150</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354232</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>variable flow chemical pump</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-077-67</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

primary culture of adult rat heart myocytes

Culture primaire de cellules adultes rongeurs cœur sont un système modèle important pour la recherche cardiovasculaire. Néanmoins, l&#39;établissement de solides, viables myocytes adultes cultivés peut être un défi technique, étape limitante de nombreux chercheurs. Ici, nous avons décrit un protocole pour obtenir un rendement élevé d&#39;adultes myocytes cardiaques de rat qui demeurent viables en culture pendant plusieurs jours. Le cœur est isolé et perfusé avec de la collagénase et la protéase sous faible teneur en Ca<sup&gt; 2 +</sup&gt; Conditions de récupérer les myocytes unique. Ca<sup&gt; 2 +</sup&gt; Tolérant cellules sont obtenues par augmentation progressive de la Ca extracellulaire<sup&gt; 2 +</supConcentration&gt; en trois étapes de lavage subséquentes. Les cellules sont filtrées, remis en suspension dans un milieu de culture, et plaquées sur les feuillets laminine couché. Myocytes cultivés obtenus en utilisant ce protocole sont viables pour un maximum de quatre jours et sont adaptés à la plupart des expériences dont l&#39;électrophysiologie, biochimie, l&#39;imagerie et de biologie moléculaire.

Watch this Scientific Journal Video about Primary Culture of Adult Rat Heart Myocytes at JoVE.com

Primary Culture of Adult Rat Heart Myocytes

Dans ce papier, nous avons décrit d&#39;une manière typique à isoler et cultiver les myocytes cardiaques de rat adulte. Collagénase et la protéase sont utilisés pour digérer et d&#39;isoler les myocytes unique. Myocytes cultivés suivre ce protocole répondent aux exigences les plus expérimenter.

Culture primaire des adultes myocytes cœur de rat

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

primary-culture-of-adult-rat-heart-myocytes

Research

JoVE Journal

Biology

20.2K vues.  Columbia University. Dans ce papier, nous avons décrit d'une manière typique à isoler et cultiver les myocytes cardiaques de rat adulte. Collagénase et la protéase sont utilisés pour digérer et d'isoler les myocytes unique. Myocytes cultivés suivre ce protocole répondent aux exigences les plus expérimenter.

Vidéo: Primary Culture of Adult Rat Heart Myocytes

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Culture organotypique de Retina lapin adulte

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Recherche

Biologie

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Implantation de l&#39;ingénierie tissulaire dans le coeur de rat

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

L&#39;isolement et la manipulation génétique des adultes pour les myocytes cardiaques imagerie confocale

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Établir des cultures primaires de fibroblastes adultes à partir de rongeurs

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Préparation de coeur de rat fortement couplées Mitochondries

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

L&#39;isolement et la culture primaire de cellules de foie de rat

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Méthodes pour l&#39;isolement, la culture et la caractérisation fonctionnelle des sinoatrial Node myocytes de souris adultes

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

Isolation simultanée de cardiomyocytes de haute qualité, de cellules endothéliales et de fibroblastes d&#39;un coeur de rat adulte

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Culture primaire de cellules Corticosurrénaliennes Rat et tests des fonctions Stéroïdogéniques

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...