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).

NOTA: O protocolo é concebido para a produção de 24 equivalentes de pele de espessura completa. Human fibroblastos dérmicos e queratinócitos epidérmicos foram isolados a partir de biópsias de pele de acordo com um protocolo publicado anteriormente. 21,22 Foi obtido consentimento informado de antemão e que o estudo foi aprovado pelo comitê de ética institucional em pesquisa humana dos Julius-Maximilians-Universidade Würzburg (votar 182 / 10). 1. Produção de componente dérmico <ol><li> Dissolve-se o colagénio com ácido acético a 0,1% para uma concentração final de 6 mg / ml. Para a solução de neutralização do gel, misturar 232,5 mL 2x DMEM, 7,5 ml de soro fetal de vitelo, 7,5 ml de 3 M de HEPES, 2,5 ml de sulfato de condroitina (5 mg / ml). Mantenha solução neutralização gel e solução de colágeno no gelo. Calcule 500 mL de gel por insert / pele equivalente. NOTA: Como a solução de neutralização gel será misturado com duas partes de solução de colágeno prepare dupla quantidade de colágeno. </li><li> Coloque 24 inserções em uma placa de 24 poços. </li><li> Ressuspender fibroblastos dérmicos humanos (HDF) em 10 ml de DMEM, centrifuga-se durante 5 min a 270 xg e contar as células. Extrai-se a quantidade necessária de HDF (5 x 10 4 culas por 500 ul de gel de colagénio) e centrifuga-se as células durante 5 min a 270 x g. </li><li> Remover o sobrenadante e ressuspender cuidadosamente o HDF em 4 ml de solução arrefecida de neutralização de gel, sem a produção de bolhas de ar. A partir deste passo certifique-se de trabalhar tão rápido quanto possível para evitar a solidificação prematura de géis de colagénio. </li><li> Ressuspender a solução com 8 ml de solução de colagénio. </li><li> Leve o colágeno-cell-mistura com um de vários passos-pipeta e preencher rapidamente 500 mL de mistura em cada inserção. Incubar os géis durante 20 minutos numa incubadora (37 ° C, 5% CO 2) cristalizar os géis. </li><li> Submergir os géis em 2 ml de Dulbecco Modified Eagle Médium (DMEM) + 10% de soro fetal de vitelo (FCS) por poçoe incubar-lo durante 24 horas numa incubadora (37 ° C, 5% CO 2). </li></ol> 2. A adição de queratinócitos epidérmicos humanos (HEK) <ol><li> Remova o meio após 24 horas. Dissolver proteína fibronectina humana com água ultrapura até uma concentração final de 50 ug / ml. Cobrir cada equivalente dérmico com 25 ml de solução de fibronectina. Incubar os géis durante 30 minutos numa incubadora (37 ° C, 5% CO 2). </li><li> Entretanto, ressuspender o hek em 10 ml de KGM-2 e definir a concentração de células para 1 x 10 6 células / ml com KGM-2 pronto + 5% FCS. Semente de 1 x 10 5 células HEK em cada gel. Incubar géis durante 45 minutos na incubadora (37 ° C, 5% CO 2) para permitir que as células a aderirem. </li><li> Submerse os géis com KGM-2 pronto + FCS a 5% e cultura los na incubadora (37 ° C, 5% CO 2). </li></ol> 3. Cultura do pleno Equivalentes da espessura da pele (FTSE) <ol><li> FTSE Cultura para 6-7 diasem concentração decrescente de FCS (5%, 2%, e 0% de FCS). Mudar a forma a cada 2-3 dias com a próxima menor concentração FCS. Para isso, remover completamente o meio e adicionar 1,6 ml de meio fresco. </li><li> No dia 7, remova o meio completamente. NOTA: Não toque na superfície do FTSE com a ponta da pipeta ao fazê-lo. </li><li> Coloque todos os inserir em um poço de uma placa de 6 bem com uma pinça estéril. Adicionar 1,5 ml de meio de transporte aéreo por poço, certifique-se para não molhar a superfície FTSE. Mudar meio cada 2-3 dias durante mais 14 dias. NOTA: O nível de enchimento do meio é até o menisco do FTSE. </li></ol> 4. Análise Histológica <ol><li> Fixação e parafina-incorporação do FTSE <ol><li> Remover o meio de cultura, transferir insertos em placas de 24 poços frescas e fixar tecidos por adição de 1,6 ml de paraformaldeído a 4% a cada poço e incuba-se os tecidos durante 2 h à TA. Cuidado! Paraformaldehyd é tóxico, manipular sob coifa. TransferênciaO FTSE a uma cassete a incorporação de tecidos. Remover fixador restante por lavagem com água e desidratar o tecido usando concentrações de etanol crescente. </li><li> Divida a pele equivalente por um corte vertical no meio. Coloque as duas peças em um molde de base de metal cheio de parafina com superfícies de corte para baixo. Adicionar a cassete de tecido na parte superior do molde como um suporte. </li><li> Corte fatias de 3-5 mm e flutuar-los em um banho de 40 ° C água para straitening, montar os slides em lâminas de microscópio adequados. Fatias secarem completamente. </li></ol></li><li> Hematoxilina e Eosina (H &amp; E) e imuno-histoquímica (IHQ) coloração <ol><li> Colocar as lâminas em prateleira e incubar durante 1 hora a 60 ° C. Certifique-se de que a parafina é derretida. Stain fatias com H &amp; E reidratadas como uma coloração visão geral padronizado. </li><li> Para a coloração com H &amp; E preparar 4x cuvetes de vidro para coloração com xilol, 3x com etanol a 96%, 2x com etanol a 70%, 1x com etanol a 50%, 4x de água desionizada, 1x hematoxilina, 1x HCl-pThanol (13,7 ml de 1 M de HCl ad 200 ml de etanol a 50%), 1x separador de água, 1x eosina (1 g eosina em 100 ml de água desionizada) e 2 x 2-propanol. </li><li> Colocar as lâminas 10 min em xilol I e ​​10 min em xilol II para Desparafinar e reidratar slides. Dip desliza 3x em etanol a 96% I, 3x em etanol a 96% II, 3x com etanol a 70% e 3 x em 50% de etanol. Gire as lâminas em água deionizada. Para diferenciar corante hematoxilina, 2x mergulho na HCl-etanol. Enxágüe em água deionizada. Para blueing, lugar desliza 5 min no separador de água. Para eosina, lugar desliza 1 min em eosina e lavar depois em água deionizada. Para a desidratação, dip slides 2x em etanol a 70%, 2x em etanol a 96% e colocá-las durante 5 min em 2-propanol I, durante 5 min em 2-propanol II, 5 min em xilol I e ​​5 min em xilol II. Depois de H &amp; E coloração, núcleos de células são coradas em azul, citoplasma e matriz extracelular estão manchadas de vermelho. </li><li> Para a recuperação antigênica, remover a parafina com xileno e reidratar fatias para a coloração. Coloque as fatiasna panela a vapor e cozinhe fatias desparafinizadas e reidratadas por 20 min em pré-aquecido 10 mM tampão citrato (pH 6). </li><li> Colocar as lâminas em tampão de lavagem (PBS tampão + 0,5% polissorbato 20) e fatias de círculo com um repelente de slides caneta stylus líquido ou diamante. </li><li> Colocar as lâminas em câmara úmida. O bloqueio da peroxidase endógena, por adição de 100 ul solução de peróxido de hidrogénio a 3% para cada fatia. Cuidado! Muito perigoso em caso de pele e dos olhos contato. Manusear com equipamentos de proteção individual. Lave as lâminas em tampão de lavagem. </li><li> Aplicar o anticorpo primário e incubar durante 1 h à TA. Lave as lâminas três vezes com tampão de lavagem. Daqui em diante, as amostras devem ser mantidas no escuro. </li><li> Use sistema de detecção biotina-estreptavidina para a detecção da ligação antigénio-anticorpo específico. </li><li> Núcleos contracorante com Hematoxilina de 1 min. Desidratar e montar slides. </li><li> Amostras de imagem usando um microscópio invertido. </li></ol></li></ol> &lt;p class = &quot;jove_title&quot;&gt; 5. Lesão por uso do dispositivo Automated Ferimento (AWD) <ol><li> Prepare o AWD por desinfecção da área de trabalho com etanol a 70% e luz ultravioleta. Certifique-se que a cabeça de perfuração do tamanho desejado (por exemplo, 1 mm), está ligado. </li><li> Coloque equivalentes de pele em áreas designadas da placa de suporte da amostra autoclavado usando uma pinça estéril. Coloque a placa de suporte à tomada de amostra abaixo da cabeça de perfuração. </li><li> Use o software de controle para definir os parâmetros ferindo da seguinte forma: a velocidade de rotação: 15.000 rpm; penetração: 1,5 milímetros; propulsão: 100 Hz. Esses parâmetros devem ser definidos para cada tipo de amostra individualmente. Transferência ferindo parâmetros para o AWD. </li><li> Comece ferindo procedimento. Retire a placa transportadora amostra da tomada. Transferência feridos FTSE volta em pastilhas usadas para a cultura usando uma pinça estéril. </li><li> Continuar com a cultura de FTSE feridos na incubadora (37 ° C, 5% CO 2) para a investigação de regeneração. Alternatively, usar os modelos para a análise histológica em qualquer momento. </li></ol>

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The authors have nothing to disclose.

Em células in vitro são normalmente expandido em culturas de células de duas dimensões, em que as células aderem a superfícies de plástico. No entanto, estas condições de cultura não reflectem as condições tridimensionais fisiológicas em que as células crescem in vivo. Sob condições tridimensionais, as células podem formar ligações célula-célula e célula-matriz naturais e migrar em três dimensões. Especialmente na cura de feridas cutâneas a semelhança da situação <e...

A pele é o maior órgão do corpo. Ela cria uma barreira entre o ambiente externo e os órgãos internos. Além disso, a pele protege o corpo contra a perda de fluido, as influências ambientais, infecções e lesões e ajuda a regular a temperatura do corpo 1. Devido à sua localização exposta, a pele é frequentemente afetada pelo trauma mecânico, térmico ou químico. Embora a pele é geralmente capaz de auto-reparo, múltiplos fatores locais, como infecção, oxigenação e suficiência venosa pode levar a cicatrização de feridas prejudicada. Cicatrização de feridas também pode ser interferido por fatores sistêmicos como a obesidade, alcoolismo, tabagismo, medicação, nutrição e doenças como a diabetes. 2 O processo de cicatrização de feridas pode ser dividida em três fases: (i) a fase inflamatória, (ii) o proliferativas e o (iii) em fase de remodelação. Após lesão da pele, um sinal complexo inicia-cascata, levando ao encerramento da ferida. 3Após um traumatismo, a ferida é sangramento e um coágulo sanguíneo é formado. Os fibroblastos passar para o coágulo de sangue e substituí-lo por um novo tecido que seja posteriormente remodelada ao longo dos anos. O entendimento atual da reparação cutânea processos biológicos subjacente é limitado. Pequenos modelos animais e de suínos têm sido usados ​​para estudar a cicatrização de feridas. No entanto, estes resultados não podem ser transferidos diretamente para os seres humanos devido a diferenças específicas de cada espécie. Em adição a estes modelos in vivo, alguns aspectos da cicatrização de feridas pode ser estudado através da simulação de uma situação de ferida através de riscar em culturas de monocamada in vitro com base em linhas celulares imortalizadas ou células primárias. 4 Estes modelos riscar são altamente padronizados, mas não suficientemente reflectir o fisiologia complexo in vivo. Além disso 5 modelos bidimensionais, tridimensionais equivalentes de pele humana foram desenvolvidas para a investigação dermatológica. A parte dérmica desses modelos são generated utilizando diversos suportes, incluindo derme decelularizados, 6 hidrogéis colágeno, 7,8 glicosaminoglicanos 9 ou materiais sintéticos. 10 Empregando estas equivalentes de pele, o papel das interações epitélio-mesenquimal 11, a re-epitelização, o crosstalk celular entre fibroblastos e queratinócitos e a influência de diferentes factores de crescimento pode ser estudada. Além disso, estes modelos são úteis para obter novos conhecimentos sobre como fibroblastos migram para a área ferida e como fatores quimiotáticos influenciar a regeneração dos tecidos 12. Não só geração do próprio modelo a cicatrização de feridas é um desafio, mas também para estabelecer uma ferida altamente padronizada em um modelo é problemático. As técnicas comuns para criar as feridas são testes de risco, 13 queimaduras, 14 de fita de abrasão, 15 lesões térmicas, 16 blisters de sucção, nitrogênio líquido 17, 18, ​​19 lasers </sup&gt; bisturis, 18 meshers 6 e punções. 20 A maioria desses métodos têm as mesmas armadilhas. Lesões implementadas manualmente são difíceis de padronizar e se reproduzir entre vários testes. O tamanho, forma e profundidade da ferida variar entre estudos e, assim, prejudicar a qualidade dos dados de pesquisa. O uso do laser para ferir a pele pode ser definido de forma relativamente fácil padronizado, mas conduz a uma situação que imita queimaduras. O calor aplicado por laser pode provocar a desnaturação da proteína, agregação de plaquetas constrição ou navios, o que pode levar ao tecido necrótico. Em uma abordagem alternativa, foi desenvolvido um dispositivo ferimento automatizado (AWD), que nos permite gerar feridas cutâneas definidas e precisas em condições estéreis. Parâmetros ferindo, como profundidade e velocidade de penetração, bem como as revoluções da cabeça de perfuração pode ser regulada. Neste estudo, nós combinamos o AWD com em casa desenvolvidos espessura completa equivalentes de pele (ftSE), que são comparáveis ​​a um protocolo publicado por Gangatirkar et al. 8 a camada dérmica da pele equivalente é composta por fibroblastos dérmicos humanos (HDF), que são incorporados em colagénio I um hidrogel. Na camada dérmica, os queratinócitos epidérmicos humanos (HEK) são semeadas. Dentro de duas semanas na interface ar-líquido da HEK construir-se uma epiderme composta de várias camadas de células vitais e um estrato córneo. Para além da geração de este modelo, este estudo mostra a utilização do AWD para criar feridas definidos e precisos em FTSE.

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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>
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			<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.

O HDF e hek isolado diferem tanto na morfologia e na expressão de marcadores típicos. O HDF mostraram morfologia típica forma de fuso, ao passo que a morfologia de hek pode ser descrito por uma morfologia de paralelepípedos. As células foram caracterizadas por coloração imuno-histoquímica (Figura 1) antes de utilizá-los para FTSE. O HDF são positivas para a vimentina (Figura 1A), um marcador para os fibroblastos. Primário HEK altamente expressar proteína diferenciação prec...

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.

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

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 visualizações.  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. 

Vídeo: 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.

A Pele Tridimensional Humanos Reconstruir Modelo: uma Ferramenta para o Estudo da Pele Normal e Progressão Melanoma

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

Bioengenharia

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.

Impressão de planar e tridimensional de tintas condutoras

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.

Investigando a separação de fluxo tridimensional induzida por um polip de dobra vocal modelo

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.

A pele completa Defeito Modelo Estudar a vascularização de Biomateriais<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.

Análise da migração celular dentro de uma matriz de colagénio tridimensional

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.

Engenharia tridimensionais tecidos epiteliais incorporado dentro Matriz Extracelular

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.

Tridimensional de impressão de materiais termoplásticos para criar automatizado bombas de seringa com controle de gabarito para aplicações microfluídicos

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.

Cultivando uma Epiderme Humana Reconstruída Tridimensional em grande escala

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.

Geração de um modelo simplificado de pele em um chip tridimensional em uma plataforma microfluida micromaquinda

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.