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: Il protocollo è progettato per la produzione di 24 intero spessore equivalenti pelle. Umano fibroblasti dermici e cheratinociti epidermici sono stati isolati da biopsie cutanee secondo un protocollo precedentemente pubblicato. 21,22 il consenso informato è stato ottenuto in anticipo e lo studio è stato approvato dal comitato etico istituzionale sulla ricerca umana della Julius-Maximilians-Universität Würzburg (voto 182 / 10). 1. Produzione di Dermal Component <ol><li> Sciogliere collagene con 0,1% di acido acetico ad una concentrazione finale di 6 mg / ml. Per la soluzione di neutralizzazione del gel, mescolare 232,5 ml 2x DMEM, siero fetale di vitello 7,5 ml, 7,5 ml 3 M HEPES, 2,5 ml condroitin solfato (5 mg / ml). Mantenere la soluzione di neutralizzazione del gel e soluzione di collagene su ghiaccio. Calcola 500 ml di gel per equivalente di inserimento / pelle. NOTA: Come la soluzione di gel di neutralizzazione sarà mescolato con due parti di soluzione di collagene prepare doppia quantità di collagene. </li><li> Mettere 24 inserisce in un piatto ben 24. </li><li> Risospendere fibroblasti dermici umani (HDF) in 10 ml DMEM, centrifugare per 5 min a 270 xg e contare le cellule. Estrarre la quantità necessaria di HDF (5 x 10 4 cellule per 500 microlitri gel di collagene) e centrifugare le cellule per 5 min a 270 x g. </li><li> Rimuovere il surnatante e con attenzione risospendere il HDF in 4 ml di soluzione raffreddata neutralizzazione gel senza produrre bolle d&#39;aria. Da questo punto assicurarsi di lavorare più velocemente possibile per evitare solidificazione prematura del gel di collagene. </li><li> Risospendere la soluzione con 8 ml di soluzione di collagene. </li><li> Prendere la cellula-miscela di collagene con un multistep-pipetta e riempire rapidamente 500 ml di miscela in ogni inserto. Incubare il gel per 20 minuti in un incubatore (37 ° C, 5% CO 2) per gelificano i gel. </li><li> Immergere il gel in ml di coltura di Dulbecco modificato (DMEM) siero 2 + 10% di vitello fetale (FCS) per pozzettoe incubare per 24 ore in un incubatore (37 ° C, 5% CO 2). </li></ol> 2. Aggiunta di Human Epidermal cheratinociti (HEK) <ol><li> Rimuovere media dopo 24 ore. Sciogliere proteina umana fibronectina con acqua ultrapura ad una concentrazione finale di 50 mg / ml. Coprire ogni cutanea equivalente con 25 ml di soluzione di fibronectina. Incubare il gel per 30 min in un incubatore (37 ° C, 5% CO 2). </li><li> Nel frattempo, risospendere il Hek in 10 ml KGM-2 e impostare concentrazione cellulare di 1 x 10 6 cellule / ml con KGM-ready 2 + 5% FCS. Seme 1 x 10 5 HEK su ogni gel. Incubare gel per 45 min in incubatore (37 ° C, 5% CO 2) per permettere alle cellule di aderire. </li><li> Immergere i gel con KGM-2 pronto + 5% FCS e cultura loro in incubatore (37 ° C, 5% CO 2). </li></ol> 3. Cultura della completa equivalenti pelle Spessore (FTSE) <ol><li> Cultura Ftse per 6-7 giorniin decrescente FCS-concentrazione (5%, 2% e 0% FCS). Cambiare la media ogni 2-3 giorni con la successiva concentrazione FCS inferiori. Per questo, rimuovere completamente il mezzo e aggiungere 1,6 ml di mezzo fresco. </li><li> Il giorno 7, rimuovere completamente media. NOTA: Non toccare la superficie FTSE con la punta della pipetta, mentre farlo. </li><li> Svolge ogni inserire in 1 pozzetto di una piastra 6 bene con pinze sterili. Aggiungere 1,5 ml di mezzo di trasporto aereo per bene, assicurarsi di non bagnare la superficie Ftse. Cambiare media ogni 2-3 giorni per altri 14 giorni. NOTA: Il livello di riempimento del mezzo è fino al menisco del FTSE. </li></ol> 4. Analisi istologica <ol><li> Fissaggio e paraffina embedding del FTSE <ol><li> Togliere terreno di coltura, di trasferire gli inserti in fresco 24 pozzetti e fissare i tessuti con l&#39;aggiunta di 1,6 ml di 4% paraformaldeide a ciascun pozzetto e incubare i tessuti per 2 ore a temperatura ambiente. Attenzione! Paraformaldehyd è tossico, manipolare sotto cappa aspirante. Trasferimentoil FTSE di una cassetta di tessuto-embedding. Eliminare le rimanenti fissativo mediante lavaggio con acqua e disidratare i tessuti con le concentrazioni ascendente di etanolo. </li><li> Dividere la equivalente pelle un taglio verticale nel mezzo. Mettere i due pezzi in uno stampo di base metallica di paraffina in, con superfici di taglio verso il basso. Aggiungere la cassetta tessuto sulla parte superiore dello stampo come supporto. </li><li> Tagliare 3-5 micron fette e li galleggiare su un bagno d&#39;acqua a 40 ° per straitening, montare scivoli su appositi vetrini da microscopio. Fette asciugare accuratamente. </li></ol></li><li> Hematoxylin &amp; eosina (H &amp; E) e immunoistochimica (IHC) colorazione <ol><li> Collocare i vetrini in rack e incubare per 1 ora a 60 ° C. Assicurarsi che la paraffina è sciolto. Stain fette con H &amp; E reidratato come una panoramica colorazione standard. </li><li> Per H &amp; E colorazione preparare 4x Cuvette colorazione vetro con xilolo, 3x con etanolo 96%, 2x con etanolo al 70%, 1x con etanolo al 50%, 4x acqua deionizzata, 1x ematossilina, 1x HCl-eTHANOL (13.7 ml 1 M HCl ad 200 ml di etanolo al 50%), 1x scheda acqua, 1x eosina (1 g eosina in 100 ml di acqua deionizzata) e 2x 2-propanolo. </li><li> Collocare i vetrini 10 min in xilolo I e 10 minuti in xilolo II Deparaffinare e reidratare le diapositive. Dip scivola 3x in etanolo 96% I, 3x in etanolo 96% II, 3x in etanolo al 70% e 3x in etanolo 50%. Ruotare i vetrini in acqua deionizzata. Per differenziare ematossilina tintura, tuffo 2x in HCl-etanolo. Risciacquare con acqua deionizzata. Per brunitura, posto scivola 5 min in acqua tab. Per eosina, posto scivola 1 min in eosina e lavare poi in acqua deionizzata. Per disidratazione, dip vetrini 2x in etanolo 70%, 2x in etanolo 96% e metterli per 5 minuti in 2-propanolo I, per 5 minuti in 2-propanolo II, 5 min in xilolo I e 5 min in xilolo II. Dopo colorazione H &amp; E, nuclei delle cellule sono colorate in blu, citoplasma e matrice extracellulare sono macchiate di rosso. </li><li> Per il recupero dell&#39;antigene, rimuovere la paraffina con xilene e reidratare fette per la colorazione. Mettere le fettein forno a vapore e cuocere fette deparaffinate e reidratati per 20 min in pre-riscaldate a 10 mM tampone citrato (pH 6). </li><li> Collocare i vetrini in tampone di lavaggio (PBS tampone + 0,5% Polysorbat 20) e fette di cerchio con un pennarello repellenti diapositiva liquido o diamante stilo. </li><li> Mettere i vetrini in una camera umida. Bloccare la perossidasi endogena, con l&#39;aggiunta di 100 microlitri 3% soluzione di perossido di idrogeno su ogni fetta. Attenzione! Molto pericoloso in caso di pelle e con gli occhi. Maneggiare con dispositivi di protezione individuale. Lavare i vetrini in tampone di lavaggio. </li><li> Applicare l&#39;anticorpo primario e incubare per 1 ora a temperatura ambiente. Lavare i vetrini tre volte con tampone di lavaggio. Da qui in poi, i campioni devono essere tenuti all&#39;oscuro. </li><li> Utilizzare sistema di rilevamento biotina-streptavidina per il rilevamento di legame specifico antigene-anticorpo. </li><li> Nuclei Controcolorare con ematossilina per 1 min. Disidratare e montare le diapositive. </li><li> Campioni immagine utilizzando un microscopio inversa. </li></ol></li></ol> &lt;p class = &quot;jove_title&quot;&gt; 5. Infortunio per l&#39;uso del dispositivo automatizzato Ferimento (AWD) <ol><li> Preparare la AWD da disinfettare l&#39;area di lavoro con il 70% di etanolo e la luce ultravioletta. Assicurarsi che la testa di foratura delle dimensioni desiderate (ad esempio, 1 mm) è attaccata. </li><li> Mettere equivalenti pelle in aree designate della piastra di supporto del campione in autoclave con pinza sterile. Posizionare la piastra di supporto del campione nella presa sotto la testa di foratura. </li><li> Utilizzare il software di controllo per impostare i parametri ferendone come segue: velocità di rotazione: 15.000 rpm; penetrazione: 1,5 millimetri; propulsione: 100 Hz. Devono essere definiti per ogni tipo di campione singolarmente Questi parametri. Trasferimento ferendo parametri AWD. </li><li> Avviare ferendo procedura. Rimuovere piastra di supporto del campione dalla presa. Trasferimento ferito Ftse nuovamente dentro inserti utilizzati per la cultura con pinza sterile. </li><li> Procedere con la cultura del FTSE feriti in incubatrice (37 ° C, 5% CO 2) per le indagini di rigenerazione. Alternativa, utilizzare i modelli per l&#39;analisi istologica in qualsiasi momento. </li></ol>

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The authors have nothing to disclose.

In cellule in vitro sono normalmente espanso in colture cellulari bidimensionali, in cui le cellule aderiscono a superfici plastiche. Tuttavia, queste condizioni di coltura non riflettono le condizioni tridimensionali fisiologiche in cui le cellule crescono in vivo. In condizioni tridimensionali, le cellule possono formare allegati cellula-cellula e cellula-matrice naturali e migrare in tre dimensioni. Soprattutto nella ferita cutanea guarigione la somiglianza della situazione in vivo...

La pelle è l&#39;organo più grande del corpo. Si crea una barriera tra l&#39;ambiente esterno e gli organi interni. Inoltre, la pelle protegge il corpo da perdita di liquidi, le influenze ambientali, ferite e infezioni e aiuta a regolare la temperatura corporea 1. Grazie alla sua posizione esposta, la pelle è spesso influenzata da traumi meccanici, termici o chimici. Anche se la pelle è generalmente in grado di auto-riparazione, molteplici fattori locali, come l&#39;infezione, l&#39;ossigenazione, e la sufficienza venosa può portare a compromissione della guarigione della ferita. La guarigione delle ferite può essere disturbato da fattori sistemici come l&#39;obesità, l&#39;alcolismo, il fumo, i farmaci, la nutrizione e le malattie come il diabete. 2 Il processo di guarigione della ferita può essere divisa in 3 fasi: (i) la fase infiammatoria, (ii) la proliferativi e (iii) fase di rimodellamento. Su lesioni alla pelle, un complesso segnale cascade inizia, che porta alla chiusura della ferita. 3Dopo l&#39;infortunio, la ferita sanguina e un coagulo di sangue si forma. I fibroblasti muovono nel coagulo di sangue e sostituirlo con nuovo tessuto che viene successivamente ristrutturato nel corso degli anni. L&#39;attuale comprensione della riparazione cutanea processi biologici di base è limitata. Modelli piccoli animali e suini sono stati utilizzati per studiare la guarigione delle ferite. Tuttavia, questi risultati non possono essere trasferiti direttamente all&#39;uomo a causa di differenze specifiche specie. Oltre a questi modelli in vivo, alcuni aspetti della guarigione delle ferite possono essere studiati simulando una situazione ferita con graffiare in vitro colture monostrato basato su linee cellulari immortalizzate o cellule primarie. 4 Questi modelli razzolare altamente standardizzati ma non sufficientemente riflettono la complesso la fisiologia in vivo. 5 Oltre ai modelli bidimensionali, tridimensionali equivalenti pelle umana sono stati sviluppati per la ricerca dermatologica. La parte cutanea di questi modelli sono generated utilizzando vari ponteggi compresi derma decellularized, 6 idrogel di collagene, glicosaminoglicani 7,8 9 o materiali sintetici. 10 Impiegando questi equivalenti pelle, il ruolo delle interazioni epiteliali-mesenchimali 11, la riepitelizzazione, la diafonia tra cellulare fibroblasti e cheratinociti e influenza dei diversi fattori di crescita può essere studiato. Inoltre, questi modelli sono utili per acquisire nuove conoscenze su come fibroblasti migrano nella zona ferita e come fattori chemiotattici influenzano la rigenerazione dei tessuti. 12 Non solo la generazione del modello guarigione della ferita in sé è impegnativo, ma anche per stabilire una ferita altamente standardizzato in un modello è problematico. Tecniche comuni per creare le ferite sono test e vinci, 13 ustioni, 14 nastro abrasione, 15 lesioni termiche, 16 blister di aspirazione, l&#39;azoto liquido 17, 18 laser, 19 </sup&gt; bisturi, 18 meshers 6 e pugni biopsia. 20 La maggior parte di questi metodi hanno le stesse insidie. Gli infortuni implementate manualmente sono difficili da standardizzare e riprodurre tra più prove. Dimensioni, forma e profondità della ferita variano tra gli studi e quindi compromettono la qualità dei dati di ricerca. L&#39;uso del laser per ferire la pelle definito può essere relativamente facile standardizzato, ma porta ad una situazione mimando ustioni. Il calore applicato dal laser può causare denaturazione delle proteine, le piastrine aggregazione o vasi costrizione, che può portare a tessuto necrotico. In un approccio alternativo, abbiamo sviluppato un dispositivo ferimento automatico (AWD), che ci permette di generare le ferite cutanee definiti e precisi in condizioni sterili. Parametri ferimento, come la profondità e la velocità di penetrazione e giri la testa di perforazione sono regolabili. In questo studio, abbiamo combinato l&#39;AWD con in casa sviluppati spessore pieno equivalenti pelle (ftSE) che sono paragonabili a un protocollo pubblicato da Gangatirkar et al. 8 Lo strato dermico dell&#39;equivalente pelle è composto di fibroblasti dermici umani (HDF), che sono incorporati in un collagene I idrogel. Sullo strato dermico, cheratinociti epidermici umani (HEK) sono teste di serie. Entro due settimane all&#39;interfaccia aria-liquido la HEK costruire una epidermide composta di vari strati di cellule vitali e un strato corneo. Oltre alla generazione di questo modello, questo studio mostra l&#39;uso del awd per creare ferite definite e precise in FTSE.

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			<td height="21" style="height:21px;width:329px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<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>
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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.

Il HDF isolata e Hek differiscono sia nella morfologia e nella espressione di marcatori tipici. HDF mostrava tipica morfologia fuso-forma, considerando morfologia Hek può essere descritto da una morfologia ciottoli. Le cellule sono state caratterizzate mediante colorazione immunoistochimica (Figura 1) prima di utilizzarli per FTSE. HDF sono positive per vimentina (Figura 1A), un marcatore per fibroblasti. Primaria HEK altamente esprimere presto proteina differenziazione citocheratina-1...

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.

Generazione di una tridimensionale a tutto spessore della pelle Equivalent 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

18.6K Views.  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. 

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensionale con risoluzione ottica Microscopia fotoacustico

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Ricerca

Bioingegneria

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Idrogeli di alginato per tridimensionale Cultura Organo di ovaie e tube

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

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.

Tridimensionale pelle umana Ricostruire Modello: uno strumento per studiare la pelle normale e progressione Melanoma

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

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.

Stampa planare e tridimensionale di inchiostri conduttivi

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.

Investigare la separazione tridimensionale del flusso indotta da un modello di polipo piega vocale

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.

Una pelle piena Difetto modello per valutare Vascolarizzazione di Biomateriali<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.

Analisi della migrazione delle cellule all&#39;interno di una tridimensionale di collagene Matrix

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

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Preparazione e analisi di<em&gt; In Vitro</em&gt; Tridimensionale carcinoma mammario mondo dei replicanti

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

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.

Tridimensionale di stampa di materiali termoplastici per creare automatizzati pompe a siringa con controllo in retroazione per applicazioni di microfluidica

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.

Coltivare un'epidermide umana ricostruita tridimensionale su larga scala

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