The authors thank the Fraunhofer ISC for the collaboration concerning the construction of the automated wounding device. The project was founded by Fraunhofer internal project &#8220;M&#228;rkte von &#220;bermorgen&#8221; (SkinHeal).

NOTE: Le protocole est conçu pour la production de 24 équivalents de pleine épaisseur de la peau. Fibroblastes dermiques et les kératinocytes épidermiques humains ont été isolés à partir de biopsies de la peau selon un protocole précédemment publié. 21,22 consentement éclairé a été obtenu au préalable et l&#39;étude a été approuvé par le comité d&#39;éthique institutionnels sur la recherche humaine de Julius-Maximilians-Université de Würzburg (voter 182 / 10). 1. Production de composants cutanée <ol><li> Dissoudre le collagène avec de l&#39;acide acétique à 0,1% à une concentration finale de 6 mg / ml. Pour la solution de neutralisation du gel, mélanger 232,5 ml 2x DMEM, 7,5 ml de sérum de veau foetal, 7,5 ml de 3 M HEPES, 2,5 ml de sulfate de chondroïtine (5 mg / ml). Conserver la solution de neutralisation de gel et de solution de collagène sur la glace. Calculer 500 pi de gel par équivalent insert / peau. NOTE: Comme la solution de gel de neutralisation sera mélangé avec deux parties d&#39;une solution de collagène prépare double du montant de collagène. </li><li> Placez 24 insertions dans une plaque à 24 puits. </li><li> Reprendre fibroblastes dermiques humains (HDF) dans 10 ml de DMEM, centrifugeuse pendant 5 min à 270 xg et compter les cellules. Extraire la quantité nécessaire de HDF (5 x 10 4 cellules par 500 pi gel de collagène) et centrifuger les cellules pendant 5 min à 270 x g. </li><li> Eliminer le surnageant et remettre en suspension avec précaution dans le HDF 4 ml refroidis solution de neutralisation du gel sans produire de bulles d&#39;air. De cette étape assurez-vous de travailler aussi vite que possible pour éviter la solidification prématurée des gels de collagène. </li><li> Remettre en suspension de la solution avec 8 ml d&#39;une solution de collagène. </li><li> Prenez le collagène cellules-mélange avec un plusieurs étapes-pipette et remplir rapidement 500 pi de mélange dans chaque insert. Incuber les gels pendant 20 minutes dans un incubateur (37 ° C, 5% CO 2) à gélifier les gels. </li><li> Immerger les gels dans 2 ml de milieu Eagle modifié par Dulbecco (DMEM) + 10% de sérum de veau foetal (FCS) par puitset incuber pendant 24 heures dans un incubateur (37 ° C, 5% CO 2). </li></ol> 2. Ajout de kératinocytes épidermiques humains (HEK) <ol><li> Retirez milieu après 24 h. Dissoudre protéine humaine de la fibronectine avec de l&#39;eau ultra-pure à une concentration finale de 50 pg / ml. Couvrir chaque derme équivalent avec 25 pi de solution de fibronectine. Incuber les gels pendant 30 minutes dans un incubateur (37 ° C, 5% CO 2). </li><li> Dans le même temps, remettre en suspension le hek dans 10 ml KGM-2 et régler la concentration de cellules de 1 x 10 6 cellules / ml avec KGM-2 prêt + 5% de FCS. Seed 1 x 10 5 Hek sur chaque gel. Incuber les gels pendant 45 minutes dans l&#39;incubateur (37 ° C, 5% CO 2) pour permettre aux cellules d&#39;adhérer. </li><li> Immerger les gels avec KGM-2 prêt + 5% de FCS et leur culture dans l&#39;incubateur (37 ° C, 5% CO 2). </li></ol> 3. Culture de équivalents plein épaisseur de la peau (FTSE) <ol><li> FTSE de Culture pour 6-7 joursen diminuant FCS-concentration (5%, 2% et 0% de FCS). Changer le support tous les 2-3 jours de la prochaine concentration inférieure FCS. Pour cela, supprimer complètement le moyen et ajouter 1,6 ml de milieu frais. </li><li> Au jour 7, supprimer complètement moyenne. REMARQUE: Ne touchez pas la surface FTSE avec le bout de la pipette en le faisant. </li><li> Lieu tous les insérer dans une puits d&#39;une plaque à 6 puits avec des pinces stériles. Ajouter 1,5 ml de milieu de transport aérien par puits, veiller à ne pas mouiller la surface FTSE. Changer le milieu tous les 2-3 jours pour 14 jours supplémentaires. NOTE: Le niveau du milieu de remplissage est au ménisque du FTSE. </li></ol> 4. Analyse histologique <ol><li> Fixation et paraffine-encastrement du FTSE <ol><li> Retirez milieu de culture, transférer inserts en plaques à 24 puits frais et fixer les tissus en ajoutant 1,6 ml de paraformaldéhyde 4% dans chaque puits et incuber les tissus pendant 2 heures à la température ambiante. Attention! Paraformaldehyde est toxique, manipuler sous une hotte. TransfertL&#39;indice FTSE une cassette de tissu-enrobage. Retirer fixatif restant par lavage avec de l&#39;eau et de déshydrater le tissu en utilisant des concentrations d&#39;éthanol ascendant. </li><li> Diviser l&#39;équivalent de peau par une coupe verticale au milieu. Placer les deux pièces dans un moule de base métallique rempli de paraffine surfaces de coupe vers le bas. Ajouter la cassette de tissus au-dessus du moule en tant que support. </li><li> Couper 3-5 um tranches et les faire flotter sur un bain d&#39;eau à 40 ° pour les opprimer, les lamelles sur des lames de microscope appropriés. Tranches sécher complètement. </li></ol></li><li> Hématoxyline &amp; éosine (H &amp; E) et immunohistochimique (IHC) coloration <ol><li> Placer les lames dans le rack et incuber pendant 1 heure à 60 ° C. Assurez-vous que la paraffine est fondue. Stain réhydraté tranches avec H &amp; E comme un aperçu coloration standardisée. </li><li> Par coloration H &amp; E préparer 4x cuvettes de coloration de verre avec du xylol, 3x avec de l&#39;éthanol à 96%, 2 fois avec de l&#39;éthanol 70%, 1 x avec de l&#39;éthanol à 50%, 4x eau désionisée, 1x hématoxyline, 1x HCl-eTHANOL (13,7 ml de HCl 1 M ad 200 ml 50% d&#39;éthanol), l&#39;eau du robinet 1x, 1x éosine (éosine 1 g dans 100 ml d&#39;eau désionisée) et 2x 2-propanol. </li><li> Placer les lames 10 min dans du xylol I et 10 min dans du xylol II Déparaffiner et réhydrater diapositives. Tremper les lames 3x dans l&#39;éthanol 96% I, 3x dans l&#39;éthanol 96% II, 3x dans l&#39;éthanol 70% et 3x dans l&#39;éthanol 50%. Tournez lames à l&#39;eau déminéralisée. Pour différencier hématoxyline colorant, 2x plongeon dans HCl-éthanol. Rincer à l&#39;eau déminéralisée. Pour bleuissement, placer les lames 5 min dans l&#39;eau du robinet. Pour éosine, le lieu glisse 1 min dans l&#39;éosine et laver ensuite dans de l&#39;eau déminéralisée. Pour la déshydratation, plaques d&#39;immersion 2x dans l&#39;éthanol 70%, 2x dans l&#39;éthanol 96% et les placer pendant 5 min dans 2-propanol I, pendant 5 min dans 2-propanol II, 5 min dans du xylol I et 5 min dans du xylol II. Après coloration H &amp; E, des noyaux cellulaires sont colorés en bleu, le cytoplasme et la matrice extracellulaire sont colorées en rouge. </li><li> Pour la récupération de l&#39;antigène, enlever la paraffine avec du xylène et de se réhydrater tranches pour la coloration. Placer les tranchesen cuiseur à vapeur et faire cuire les tranches déparaffinées et réhydratées pendant 20 min dans préchauffées tampon citrate 10 mM (pH 6). </li><li> Placer les lames dans du tampon de lavage (tampon PBS + 0,5% Polysorbat 20) et les tranches de cercle avec un marqueur liquide répulsif de glissement ou de diamant stylet. </li><li> Placer les lames dans une chambre humide. Bloquer la peroxydase endogène, par addition de 100 ul de solution à 3% de peroxyde d&#39;hydrogène sur chaque tranche. Attention! Très dangereux en cas de contact avec la peau et les yeux. Manipuler avec équipement de protection individuelle. Laver les lames dans du tampon de lavage. </li><li> Appliquer un anticorps primaire et incuber pendant 1 heure à température ambiante. Laver les lames trois fois avec du tampon de lavage. A partir de là, les échantillons doivent être conservés dans l&#39;obscurité. </li><li> Utiliser le système de détection de la biotine-streptavidine pour la détection de la liaison antigène-anticorps spécifique. </li><li> Noyaux Counterstain à l&#39;hématoxyline pendant 1 min. Déshydrater et monter diapositives. </li><li> échantillons de l&#39;image en utilisant un microscope inversé. </li></ol></li></ol> &lt;p class = &quot;jove_title&quot;&gt; 5. Blessure par l&#39;utilisation du dispositif automatisé Blessant (AWD) <ol><li> Préparer le AWD en désinfectant la zone de travail avec 70% d&#39;éthanol et la lumière ultraviolette. Assurez-vous que la tête de forage de la taille désirée (par exemple, 1 mm) est fixé. </li><li> Placez équivalents de peau dans les régions désignées de la plaque de support de l&#39;échantillon autoclave en utilisant des pinces stériles. Placer la plaque de support d&#39;échantillon dans la douille au-dessous de la tête de forage. </li><li> Utilisez le logiciel de commande pour régler les paramètres blessant comme suit: vitesse de rotation: 15000 tours par minute; pénétration: 1,5 mm; propulsion: 100 Hz. Ces paramètres doivent être définis pour chaque type d&#39;échantillon individuellement. Transfert blessant paramètres au stade AWD. </li><li> Procédure de départ blessant. Retirer échantillon plaque de support de la prise. Transfert blessé FTSE retour dans des inserts utilisés pour la culture à l&#39;aide de pinces stériles. </li><li> Procéder à la culture de FTSE blessés dans l&#39;incubateur (37 ° C, 5% de CO 2) pour l&#39;étude de la régénération. Alvariante, utiliser les modèles pour l&#39;analyse histologique à tout moment. </li></ol>

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The authors have nothing to disclose.

Dans les cellules in vitro sont généralement étendu dans des cultures cellulaires bidimensionnelles, dans lequel les cellules adhèrent aux surfaces plastiques. Cependant, ces conditions de culture ne reflètent pas les conditions physiologiques en trois dimensions dans laquelle les cellules se développent in vivo. Dans des conditions tridimensionnelles, les cellules peuvent former des attachements cellule-cellule et cellule-matrice naturelles et de migrer dans les trois dimensions. Surtou...

La peau est le plus grand organe du corps. Il crée une barrière entre l&#39;environnement extérieur et les organes internes. En outre, la peau protège le corps contre la perte de liquide, influences de l&#39;environnement, des blessures et des infections et aide à réguler la température corporelle 1. Grâce à sa situation exposée, la peau est souvent affectée par un traumatisme mécanique, thermique ou chimique. Bien que la peau est généralement capable d&#39;auto-réparation, de multiples facteurs locaux tels que l&#39;infection, l&#39;oxygénation, et la suffisance veineuse peuvent conduire à la guérison des plaies. La cicatrisation des plaies peut également être perturbé par des facteurs systémiques que l&#39;obésité, l&#39;alcoolisme, le tabagisme, les médicaments, la nutrition et les maladies telles que le diabète. 2 Le processus de la cicatrisation peut être divisé en trois phases: (i) la phase inflammatoire, (ii) la prolifération et de la (iii) la phase de remodelage. Après une blessure de la peau, une cascade complexe de signal commence, conduisant à la fermeture de la plaie. 3Après une blessure, la plaie saigne et un caillot de sang se forme. Fibroblastes se déplacent dans le caillot de sang et le remplacer par un nouveau tissu qui est ensuite remodelé au fil des ans. La compréhension actuelle de la réparation cutanée processus biologiques sous-jacent est limitée. Petits modèles animaux et de porcs ont été utilisées pour étudier la cicatrisation des plaies. Cependant, ces résultats ne peuvent être directement transmises à l&#39;homme en raison des différences spécifiques à l&#39;espèce. En plus de ces modèles in vivo, certains aspects de la cicatrisation des plaies peuvent être étudiés en simulant une situation de la plaie par l&#39;intermédiaire de rayer in vitro des cultures monocouches à base sur des lignées cellulaires immortalisées ou des cellules primaires. 4 Ces modèles de grattage sont très normalisées, mais ne reflètent pas suffisamment la la physiologie complexe in vivo. 5 Outre les modèles en deux dimensions, les équivalents en trois dimensions peau humaine ont été développés pour la recherche dermatologique. La partie cutanée de ces modèles sont geexonéré en utilisant divers échafaudages comprenant derme décellularisées, 6 hydrogels de collagène, 7,8 glycosaminoglycanes 9 ou de matériaux synthétiques. 10 L&#39;utilisation de ces équivalents de peau, le rôle des interactions épithélium-mésenchyme 11, la ré-épithélialisation, la diaphonie cellulaire entre les fibroblastes et les kératinocytes et la influence de différents facteurs de croissance peut être étudiée. En outre, ces modèles sont utiles pour acquérir de nouvelles connaissances sur la façon dont les fibroblastes migrent dans la zone blessés et comment les facteurs chimiotactique influencent la régénération des tissus. 12 Non seulement la génération du modèle de cicatrisation de la plaie elle-même est difficile, mais également pour établir une plaie hautement standardisée en un modèle est problématique. Techniques communes visant à créer des blessures sont des tests à gratter, 13, 14 brûlures bande abrasion, 15 blessure thermique, 16 ampoules d&#39;aspiration, de l&#39;azote liquide 17, 18, ​​19 lasers </sup&gt; scalpels, 18 mailleurs 6 et poinçons de biopsie. 20 La plupart de ces méthodes ont les mêmes pièges. Blessures mises en œuvre manuellement sont difficiles à normaliser et à reproduire entre plusieurs tests. Taille, forme et la profondeur de la plaie varient entre les études et donc nuisent à la qualité des données de recherche. L&#39;utilisation de laser pour blessure défini de la peau peut être relativement facile à normaliser mais conduit à une situation imitant des brûlures. La chaleur appliquée par laser peut provoquer la dénaturation des protéines, l&#39;agrégation des plaquettes ou des navires constriction, ce qui peut conduire à des tissus nécrosés. Dans une autre approche, nous avons développé un dispositif automatisé de blessure (AWD), ce qui nous permet de générer plaies cutanées définis et précis dans des conditions stériles. Blessant paramètres, comme la profondeur et la vitesse de pénétration ainsi que les révolutions de la tête de forage peuvent être réglementés. Dans cette étude, nous avons combiné l&#39;AWD avec la pleine épaisseur équivalents développés en interne de la peau (ftSE) qui sont comparables à un protocole publié par Gangatirkar et al. 8 La couche dermique de l&#39;équivalent de peau est constitué de fibroblastes dermiques humains (HDF), qui sont noyés dans un collagène I hydrogel. Sur la couche dermique, kératinocytes épidermiques humains (HEK) sont ensemencées. Dans les deux semaines à l&#39;interface air-liquide du hek accumuler un épiderme composé de plusieurs couches de cellules vitales et un stratum corneum. Outre la génération de ce modèle, cette étude montre l&#39;utilisation de la traction intégrale pour créer blessures bien précises à FTSE.

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			<td height="21" style="height:21px;width:329px;">Name of Material/ Equipment</td>
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			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:329px;">Trypsin EDTA (1:250) 0.5 % in DPBS&#160;</td>
			<td style="width:135px;">PAA</td>
			<td style="width:119px;">L11-003</td>
			<td align="right">0.05%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Collagen (6 mg/ml in 0,1 % acetic acid)</td>
			<td style="width:135px;">Produced in house</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fibronectin Human Protein, Plasma (50 &#181;g/ml)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">33016-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Inserts</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">140627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">6 well plate&#160;</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">140685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">24 well plate</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">142485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Microscope slides</td>
			<td style="width:135px;">R. Langenbrinck</td>
			<td style="width:119px;">03-0070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fibroblasts culturing (500 ml):</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM, high glucose</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>89% (445 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal bovine serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>10% (50 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinozyten culture medium (500 ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-20111</td>
			<td>89% (445 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2 SupplementPack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Gel neutralization solution (250 ml):</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Dulbecco&#8217;s Modified Eagle Medium, high Glucose Powder with L-Glutamine</td>
			<td style="width:135px;">PAA</td>
			<td style="width:119px;">G0001,3010</td>
			<td>93% (232,5 ml)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Chondroitin sulfate sodium salt from shark cartilage</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">C4384-1g</td>
			<td>1% (2,5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal bovine serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>3% (7,5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">HEPES</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:119px;">H3375-1kg</td>
			<td>3% (7,5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin model submers medium (500ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-20111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2 SupplementPack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal calf serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>5%-2% (25 ml-10 ml)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin model air-liquid interface medium (500 ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Keratinocyte Growth Medium 2 Supplement Pack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td>adding only supplements: Insulin, Hydrocortisone, Epinephrine, Transferrin, CaCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:329px;">CaCl2 (300 mM)</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:119px;">C7902-500g</td>
			<td>&#160;0,62% (3,1 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Histology:</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">IHC-Kit DCS SuperVision 2 HRP</td>
			<td style="width:135px;">DCS</td>
			<td style="width:119px;">PD000KIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Vimentin antibody</td>
			<td style="width:135px;">Abcam</td>
			<td>ab92547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CK14 antibody</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td>HPA023040-100&#181;l</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CK10 antibody</td>
			<td style="width:135px;">Dako</td>
			<td style="width:119px;">M7002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Filaggrin antibody</td>
			<td style="width:135px;">Abcam</td>
			<td>ab81468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">H&#38;E staining</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Mayer&#180;s Haemalaun</td>
			<td style="width:135px;">AppliChem</td>
			<td style="width:119px;">A0884,2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Xylol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>296325-4X2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ethanol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>32205-4X2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">HCl</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>H1758-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Eosin</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>E4009-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2-Propanolol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>I9516-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Mounting Medium</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>M1289-10ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of a three-dimensional full thickness skin equivalent and automated wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

La isolée HDF et Hek diffèrent à la fois dans la morphologie et de l&#39;expression de marqueurs typiques. HDF a montré la morphologie typique broche en forme, alors que la morphologie de Hek peut être décrit par une morphologie pavée. Les cellules ont été caractérisées par une coloration immunohistochimique (Figure 1) avant de les utiliser pour FTSE. Le HDF sont positives pour la vimentine (Figure 1A), un marqueur de fibroblastes. HEK primaire exprimer très précoce de diff...

Watch this Scientific Journal Video about Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding at JoVE.com

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Génération d&#39;une épaisseur en trois dimensions équivalent de peau et automatisé Blessant

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

generation-three-dimensional-full-thickness-skin-equivalent-automated

Research

JoVE Journal

Bioengineering

18.4K vues.  University Hospital Würzburg. The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility. 

Vidéo: Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

La peau Three-Dimensional Human Reconstruire Modèle: un outil pour étudier la peau normale et la progression du mélanome

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Recherche

Bio-ingénierie

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impression Planar et en trois dimensions des encres conductrices

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Étude de la séparation d’écoulement tridimensionnelle induite par un polype de pli vocal modèle

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Une peau pleine de défauts modèle pour évaluer la vascularisation des biomatériaux<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Analyse de la migration cellulaire dans un tridimensionnelle Matrice Collagène

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ingénierie en trois dimensions des tissus épithéliaux incorporés au sein de la matrice extracellulaire

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

En trois dimensions d’impression de matériaux thermoplastiques pour créer automatisé pompes à seringue avec commande de rétroaction pour des Applications de microfluidique

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

Cultiver un épiderme humain reconstruit tridimensionnel à grande échelle

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

Génération d’un modèle tridimensionnel simplifié de peau sur puce dans une plate-forme microfluidique micro-usinée

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.