Cette recherche est soutenue par le Programme CounterACT, National Institutes of Health (NIH), Bureau du Directeur, et l&#39;Institut national des sciences de la santé environnementale (NIEHS) Nombre Grant U54 ES015678 (MTE). SA est également soutenue par l&#39;hôpital pour enfants de Colorado / Colorado School of Mines Collaboration Award pilote # G0100394 et Colorado Hospital Research Institute Award pilote n ° de G0100471 pour enfants.

1. Cultures Rat cardiomyocytes <ol><li> Toutes les expériences ont été réalisées selon des protocoles approuvés par le soin des animaux et l&#39;utilisation comité institutionnel, IACUC. </li><li> Obtenir des cardiomyocytes de rats du cœur (ventricules) de rats mâles (240-260 g) en utilisant des méthodes décrites précédemment 4. En bref, anesthésier les animaux en utilisant une injection intrapéritonéale de pentobarbital (100 mg / kg; confirmer l&#39;anesthésie par la méthode de pincement de l&#39;orteil), puis retirez le cœur en 10,0 ml, 1 mM de Ca 2 + contenant un tampon Krebs Ringer, pH 7,4. </li><li> Rincer les coeurs 5-6x pour enlever le sang et ensuite passer à un + tampon Krebs Ringer Ca 2 gratuit contenant 0,02% et 0,06% protéase collagénase A (5,0 ml / coeur). Incuber les coeurs dans cette solution pendant 10 à 15 min à 37 ° C avec agitation occasionnelle. </li><li> Après 10-15 minutes, laver la solution enzymatique avec un tampon de Ca 2 + libre Krebs Ringer à 5 min de plus. Libérer les cellules du tissu mou nousment une pipette de 25 ml à la pipette la suspension de haut en bas plusieurs fois. </li><li> Séparer les cellules du tissu par filtration à travers une maille de nylon de 70 um et leur permettre de s&#39;installer dans Ringer Krebs contenant 0,1 mM de Ca 2 +. Suspendre le culot cellulaire dans 1,0 ml de tampon Krebs Ringer contenant 0,2 mM de Ca 2 +. </li><li> Superposer soigneusement sur 5,0 ml de 60 pg / ml d&#39;albumine de sérum bovin, BSA, 15 ml dans des tubes de centrifugation, afin de séparer les cardiomyocytes à partir de nonmyocytes et leur permettre de se déposer pendant 30 minutes. Les cardiomyocytes sont plus lourdes et se déplacent vers le fond du tube. Retirez soigneusement les cellules surnageant et les médias. Répétez cette étape une fois pour purifier les cellules. </li><li> Une transition graduelle de lavage (dans les étapes à l&#39;aide de 0,25 mM, 0,5 mM, 0,75 mM, et mM de Ca 1,0 2 tampon + contenant) les myocytes ventriculaires / cardiomyocytes pour mM de Ca 2 + contenant du tampon 1,0 et remises en suspension dans du milieu ACCT constitué de milieu Eagle modifié par Dulbecco , DMEM containing de 2 mg / ml de BSA, 2 mM de L-carnitine, la créatine 5 mM, 5 mM de taurine, 100 UI / ml de pénicilline et 100 pg / ml de streptomycine. </li><li> Plaquer les cardiomyocytes dans un milieu ACCT à une densité de 100 à 150 cellules / mm 2 sur 100 mm ou 35 mm revêtues de laminine des boîtes de culture en plastique ou des lamelles couvre-objet 40 x 22 mm de verre pré-enduites avec de la laminine (1 mg / cm 2). Après 1 heure, laver la vaisselle avec 2,0 ml ACCT pour éliminer les cellules qui ne sont pas attachés. Ajouter 10,0 ml de milieu frais et incuber les cellules. </li></ol> 2. Interface air-liquide différencié (ALI) culture de cellules humaines Airway basales <ol><li> Obtention de tissus humains trachéo-bronchique de recherche sur les maladies d&#39;échange national en vertu de l&#39;Institutional Review Board protocoles approuvés. récolte cellulaire et effectuer la culture en utilisant une procédure de 5,6 publié. Cette procédure utilise des cultures de cellules car ils sont un modèle précis pour l&#39;exposition par inhalation humaine à TIC, cependant, si nécessaire, on peut isoler et cultiver à ALI moutiliser et / ou de cellules épithéliales des voies aériennes chez le rat 7,8. </li><li> En bref, faire de petits morceaux (~ ¼ pouce) du tissu après avoir retiré tous les noeuds lymphatiques et des tissus conjonctifs. Laver le tissu plusieurs fois dans une solution de Ringer lactate. Ajouter tissus dans un tube conique de 50 ml et ajouter une solution de protease (1% Protease/0.01% de la DNase dans un milieu minimal essentiel, MEM, tissu de rapport de fluide (01:10)). </li><li> Agitez le tissu à 4 ° C pendant une nuit. Mettre fin à la dissociation du tissu par l&#39;addition de sérum bovin fœtal à 10%. Racler la surface de l&#39;épithélium avec un scalpel chirurgical et recueillir les cellules par centrifugation (2000 rpm pendant 10 min). </li><li> Cellules de la plaque au passage un sur snapwells revêtus de collagène dans un milieu spécial ALI préparés comme décrit en détail par Foucher et al 9. 5 jours de culture avant de changer d&#39;interface air-liquide (ALI) en supprimant les médias apical. </li><li> Alimenter les cellules en utilisant les médias ALI tous les deux jours et permettent aux cellules de se différencier pour 2-3 semaines supplémentaires (observer de nombreux cils battant et la sécrétion de mucus) avant d&#39;effectuer l&#39;exposition. Laver la surface apicale avec du PBS chaud ainsi que des changements dans les médias. </li></ol> 3. Chlore exposition <ol><li> Contenir le système d&#39;exposition au chlore (CES) dans une hotte chimique qualifié avec une vitesse nominale de fonctionnement de 100 pieds par minute qui fournit l&#39;enceinte de confinement secondaire nécessaire pour éviter l&#39;exposition du personnel en cas de fuites accidentelles de chlore. </li><li> Faire fonctionner le système sous une légère pression positive (0,5 pouces d&#39;eau). L&#39;air sec est amené à 15 L / min et à un niveau approprié de chlore est introduit pour atteindre la concentration désirée en chlore final. Les chambres disposent d&#39;un couvercle de verrouillage avec 4 mini serrures de BCU et une dureté joint en silicone bas pour assurer l&#39;étanchéité de pression. </li><li> Fournir un mélange Cl 2 par un contrôleur de débit massique. La CES utilise une bouteille de gaz comprimé contenant 1,0% de Cl 2 dans de l&#39;azote sec. </li><li> Réguler le débit d&#39;air de dilution à l&#39;aide duconçu sur mesure panneau de commande et de réglementer de façon similaire la concentration Cl 2 livré aux chambres d&#39;exposition. Une pompe d&#39;échantillonnage de faible volume extrait de l&#39;échappement à partir des chambres dans un analyseur de chlore pour surveiller les concentrations qui sont ensuite enregistrées sur un enregistreur de données connecté à l&#39;analyseur. </li><li> Mesurer les vitesses d&#39;écoulement à l&#39;intérieur des chambres, avant l&#39;exposition à l&#39;aide d&#39;un débitmètre pour assurer les frais de port et d&#39;échappement égales. </li><li> Préparer les cultures de cellules d&#39;exposition en enlevant les médias surnageant et ajouter du milieu frais (médias basolatérales dans les cultures ALI). Prétraitements éventuels avec les agents pourraient être effectuées à cette époque. </li><li> Exposer les cultures ALI cellules épithéliales des voies aériennes à Cl 2 gaz (50, 100 ou 300 ppm pendant 30 min) dans les deux chambres étanches polysulfone de confinement biologique. Les cardiomyocytes (cultures submergées ou sur les membranes confluentes) sont exposées à 50 ou 100 ppm de Cl 2 pendant 15 min. </li><li> Après l&#39;exposition vider les chambres à air jusqu&#39;à ce que le Cl 2 niveau tombe en dessous de 1 ppm, et peut être ouverte en toute sécurité pour éliminer les cellules (à moins de 5 min). </li></ol> 4. Transepitheliale résistance électrique (TER) Mesure <ol><li> Mesurer le TER de l&#39;air liquide interface, ALI, cultures en utilisant une voltohmmeter épithéliale avec une paire de chlorure électrodes &quot;baguettes&quot; argent. </li><li> Equilibrer l&#39;électrode de baguettes dans le ALI médias 15 min avant utilisation. </li><li> Ajouter un média chaud au apicale (1,0 ml) et basolatéral (2,0 ml) et mesurer la surface TER en utilisant les électrodes de baguettes de la voltohmmeter. </li><li> Trempez le bras le plus court de l&#39;électrode dans les médias apical et le bras le plus long dans les médias basolatérales. Cliquez sur le bouton «mesure» sur la voltohmmeter pour évaluer la résistance électrique. </li><li> Le voltohmmeter a la possibilité de mesurer ohms ou k ohms. Soustraire la résistance à travers un support de culture acellulaire à partir de la résistance mesurée aux bornes de chaque couche de cellules pour donner le transrésistance épithéliale (TER). </li></ol> 5. Caspase Mesure <ol><li> Ajouter des milieux frais ALI (2,0 ml) à la post-exposition de la surface basolatérale et incuber la cellule à la température ambiante. Recueillir milieu surnageant à 4 et 24 heures. </li><li> Mesurer la caspase 3/7 activité dans les surnageants de média en utilisant une caspase commercial 3/7 de la trousse de dosage. </li></ol> 6. Western Blot et Immunocytochemistry <ol><li> Effectuer des transferts Western utilisant des lysats cellulaires comme décrit précédemment 10. Suspendre le lysat de protéines (20 pg) dans 5x tampon réduite de l&#39;échantillon et faire bouillir pendant 5 min. Soumettre le lysat de protéine à une SDS-PAGE (4-15%) et de transférer les protéines séparées sur une membrane de nitrocellulose par transfert électrophorétique. </li><li> Bloquer la liaison non spécifique par incubation de la membrane avec 5% de lait dans du tampon de lavage (PBS + 0,1% de détergent) et de la sonde les membranes avec des anticorps primaires contre l&#39;actine ou SERCA2 sarcomère à 1:1000 dilution, pendant une nuit à 4 ° C. Ensuite, laver et incuber les membranes avec les anticorps secondaires conjugués à la peroxydase et de développer respectifs pour la détection comme décrit ci-dessus 10 en utilisant le kit de détection de la peroxydase du commerce. </li><li> Par immunocytochimie traitent des cellules vivantes cultivées sur des inserts ou des lamelles couvre-objet en verre dans des plaques de 6 puits avec 0,4% de Triton-X-100 dans du tampon de citrate de sodium à 10 mM pendant 20 min après le rinçage avec du PBS. </li><li> Bloquer la liaison non spécifique en traitant les cellules avec du sérum d&#39;âne à 5% pendant 20 min, et ensuite incuber les cellules avec de l&#39;IgG non spécifique ou spécifique des anticorps primaires individuels à l&#39;actine sarcomère ou Ki-67. </li><li> Laver les cellules avec du PBS et incubation avec des anticorps secondaires conjugués fluorescents pour détecter l&#39;anticorps primaire et un colorant nucléaire (1 ug / ml de DAPI). </li><li> Laver avec du PBS et monter des lamelles en utilisant un milieu de montage commerciales. </li><li> Visualisez la coloration en utilisant un microscope à fluorescence. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Le type le plus commun des expositions toxiques aigus se produit lorsque l&#39;on respire un produit chimique toxique dans les poumons. Ces produits chimiques peuvent aussi être prises rapidement dans la circulation sanguine et peuvent avoir un impact d&#39;autres organes tels que le cerveau et le cœur. Toxicité à l&#39;inhalation de divers agents utilisant des modèles animaux sont étudiés et largement rapporté, mais les mécanismes sont moins bien compris. Il s&#39;agit d&#39;un obstacle majeur dans le dévelop...

L&#39;exposition à des produits chimiques toxiques inhalés (CIT) / gaz comme le chlore (Cl 2) demeure un problème de santé en cours dans les expositions accidentelles ainsi que dans leur utilisation potentielle comme un agent de menace chimique. Bien que les poumons sont la cible principale, les organes tels que le coeur et le cerveau sont également touchés 1-3. Modèles in vivo sont généralement utilisés pour les tests de toxicité de TIC, mais des essais in vitro pour l&#39;évaluation de la toxicité est plus simple, plus rapide et plus rentable. En modèles in vitro permettent également une enquête approfondie des interactions agent de cellules qui peuvent être difficiles à évaluer in vivo. Ces systèmes in vitro dans l&#39;exposition sont rares et, en outre, dans certains modèles classiques où les agents toxiques sont ajoutées au milieu de culture dans lequel les cellules sont immergées, les propriétés des agents peuvent changer en raison des interactions et liaison aux composants dans le milieu. Dans de tels scénarios cellulaires des systèmes de culture tels que l&#39;interface air-liquide (ALI) des cultures de cellules primaires épithéliales des voies respiratoires humaines, proposés ici, qui peuvent être directement exposés à des agents gazeux pourrait être prometteur. Les cellules épithéliales qui tapissent les voies respiratoires sont les premières lignes de défense contre les produits chimiques toxiques inhalés. L&#39;épithélium des voies respiratoires humaine constitue une barrière physique entre la lumière et les cellules sous-jacentes dans le poumon et participe à la réaction du poumon. Il produit un certain nombre de cytokines et d&#39;autres agents pro-et anti-inflammatoires ainsi que sécrète le liquide de surface mucus / des voies respiratoires (ASL) recouvrant l&#39;épithélium. Une des limitations à immergé conventionnelle dans les systèmes de culture in vitro est également que l&#39;ASL et de mucus qui couvre la surface épithéliale est enlevé ou dilués. Cela ne reflète pas l&#39;état physiologique des cellules épithéliales pulmonaires qui sont exposés à l&#39;air. Ainsi, un idéal dans le système in vitro pour les tests de toxicité TIC devrait reproduire cette architecture. Il ya un grand intérêt dans le développement rapide de dépistage méthodes qui prédisent la toxicité in vivo. Les cellules épithéliales cultivées à l&#39;ALI différencier et avoir des structures et des fonctions bien différenciées par rapport aux cellules cultivées submergés et servir un modèle supérieur des voies respiratoires. Dans cette étude, nous décrivons l&#39;utilisation de l&#39;air liquide interface culture de voies respiratoires humaines (trachéo-bronchique) des cellules épithéliales pour tester toxique par inhalation de gaz toxiques et de le comparer avec une culture cellulaire immergé de cardiomyocytes, donc étudier une autre cible importante de la toxicité.

<table border="0" cellpadding="0" cellspacing="0" width="1128">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Name</td>
			<td style="width:329px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Rats</td>
			<td style="width:329px;">Harlan Laboratories</td>
			<td>Sprague-Dawley&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Pentobarbital</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P3761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Chlorine</td>
			<td style="width:329px;">AirGas, Inc</td>
			<td style="width:147px;">X02NI99CP163LS1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Caspase 3/7 kit&#160;</td>
			<td style="width:329px;">Promega</td>
			<td style="width:147px;">G8091</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Epithelial voltohmmeter and chopstick electrode</td>
			<td style="width:329px;">World Precision Instruments</td>
			<td style="width:147px;">EVOM and STX2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Snapwell inserts</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">07-200-708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">70 micron nylon cell strainer</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">#352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysulfone biocontainment chambers&#160;</td>
			<td style="width:329px;">BCU, Allentown Cage Equipment</td>
			<td style="width:147px;">BCU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:329px;">Life technologies</td>
			<td style="width:147px;">12491-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sarcomeric actin antibody</td>
			<td style="width:329px;">Abcam Cambridge, MA</td>
			<td style="width:147px;">ab28052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SERCA2 antibody</td>
			<td style="width:329px;">&#160;Affinity Bioreagents, Golden, CO</td>
			<td style="width:147px;">MA3-9191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ki-67 antibody</td>
			<td style="width:329px;">&#160;Dako, Carpinteria, CA</td>
			<td style="width:147px;">M7248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-488-conjugated secondary antibody</td>
			<td style="width:329px;">&#160;Invitrogen, Grand Island, NY</td>
			<td style="width:147px;">A11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">BSA</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Carnitine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Taurine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Creatinine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Krebs Ringer Buffer</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">K4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Protease</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagenase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; DNAase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Lactated Ringer solution</td>
			<td style="width:329px;">Abott Laboratories</td>
			<td style="width:147px;">7953</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Donkey serum</td>
			<td style="width:329px;">Fisher Scientific</td>
			<td style="width:147px;">017-000-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PBS, phosphate buffered saline</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">4-15% SDS-PAGE gels</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">456-1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Nitrocellulose membrane</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">162-0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Dergent, Tween&#160;</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Peroxidase detection kit</td>
			<td style="width:329px;">Pierce</td>
			<td align="right" style="width:147px;">3402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">DAPI</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Mounting media, Fluormount G</td>
			<td style="width:329px;">eBiosciences</td>
			<td style="width:147px;">00-4958-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium citrate</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">71497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MEM</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">M8028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Laminin</td>
			<td style="width:329px;">BD biosciences</td>
			<td style="width:147px;">354259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Penicillin/Streptomycin</td>
			<td style="width:329px;">Life Technologies</td>
			<td style="width:147px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FBS</td>
			<td style="width:329px;">Gibco</td>
			<td style="width:147px;">200-6140AJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cell culture model for toxic inhaled chemical testing

Les cultures cellulaires sont indispensables pour développer et d&#39;étudier l&#39;efficacité d&#39;agents thérapeutiques, en vue de leur utilisation dans des modèles animaux. Nous avons la capacité unique de modéliser les cellules de l&#39;épithélium respiratoire humain et le muscle cardiaque bien différenciées. Cela pourrait être un outil précieux pour étudier les effets délétères des produits chimiques toxiques inhalés, tels que le chlore, qui peuvent normalement interagir avec les surfaces cellulaires, et former divers sous-produits lors de la réaction avec de l&#39;eau, et la limitation de leurs effets dans des cultures submergées. Notre modèle en utilisant des cultures de cellules épithéliales des voies aériennes humaines bien différenciés à l&#39;interface air-liqiuid contourne cette limitation ainsi que fournit l&#39;occasion d&#39;évaluer les mécanismes critiques de la toxicité des produits chimiques toxiques inhalés potentiels. Nous décrivons perte accrue de l&#39;intégrité de la membrane, la caspase libération et la mort sur chimique toxique par inhalation tels que l&#39;exposition au chlore. Dans cet article, nous proposons des méthodes pour modéliser l&#39;exposition au chlore dans le cœur des mammifères et des voies respiratoires épithéliales caunes de la culture et des tests simples pour évaluer son effet sur ces types cellulaires.

Tige principale cardiomyocytes forme attachent sur ​​des matrices de laminine et se propagent et se différencient en cultures confluentes (figure 1A et son encart). Ces cellules ont été en outre caractérisés sur la base de l&#39;actine et de l&#39;expression de sarcomère SERCA2 (Figures 1B et 1C). cardiomyocytes de rat sont très sensibles à la toxicité du chlore en tant que 15 minutes d&#39;exposition à 100 ppm de chlore a causé des arrondissement des cel...

Watch this Scientific Journal Video about In vitro Cell Culture Model for Toxic Inhaled Chemical Testing at JoVE.com

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Ce protocole est conçu pour démontrer Procédé d&#39;exposition de cultures cellulaires à des produits chimiques toxiques inhalés. Exposition des différenciée interface air-liquide (ALI) des cultures de cellules épithéliales des voies aériennes fournit un modèle unique de l&#39;exposition des voies respiratoires à des gaz toxiques tels que le chlore. Dans ce manuscrit, nous décrivons effet de l&#39;exposition au chlore sur les cultures à l&#39;interface air-liquide de cellules épithéliales et de la culture submergée de cardiomyocytes. In vitro des systèmes d&#39;exposition permettent études mécanistiques importantes pour évaluer les voies qui pourraient ensuite être utilisés pour développer de nouveaux agents thérapeutiques.

Cellulaire in vitro modèle de culture pour le contrôle des produits chimiques toxiques inhalés

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

in-vitro-cell-culture-model-for-toxic-inhaled-chemical-testing

Research

JoVE Journal

Bioengineering

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

10.6K vues.  University of Colorado.  Ce protocole est conçu pour démontrer Procédé d'exposition de cultures cellulaires à des produits chimiques toxiques inhalés. Exposition des différenciée interface air-liquide (ALI) des cultures de cellules épithéliales des voies aériennes fournit un modèle unique de l'exposition des voies respiratoires à des gaz toxiques tels que le chlore. Dans ce manuscrit, nous décrivons effet de l'exposition au chlore sur les cultures à l'interface air...

Vidéo: In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositif microfluidique pour Recréer un microenvironnement des tumeurs<em&gt; In vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Recherche

Bio-ingénierie

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Polymères à mémoire de forme pour la Culture cellule active

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Autologue de cellules progénitrices endothéliales-ensemencement de la technologie et les tests de biocompatibilité pour les appareils de cardio-vasculaires dans le modèle des grands animaux

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotation systèmes de culture cellulaire de la culture de cellules humaines: les cellules du trophoblaste humain comme modèle

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritation des yeux Test (IET) pour l&#39;identification des dangers des produits chimiques irritants oculaires utilisant épithélial (RHCE) Tissue modèle reconstruit humain Cornea-like

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formation et Patterning Light-médiée hydrogels pour la culture cellulaire Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In vitro</em&gt; Orgue Culture Modèle de la murine disque intervertébral

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Méthodologie pour la chimie biomimétique Neuromodulation des rétines de Rat avec le neurotransmetteur Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Un modèle de culture matrix-adipocyte extracellulaire humaine 3D pour étudier matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...