Ringraziamo Deborah Studer per l&#39;aiuto con il bioprinter. Il lavoro è stato finanziato dal Programma dell&#39;Unione Europea Settimo programma quadro (FP7/2007-2013) convenzione di sovvenzione n ° NMP4-SL-2009-229.292.

1. Preparazione della Soluzione Poloxamer 407 Se disponibile, eseguire la preparazione della soluzione polossalene in una camera fredda (4 ° C). Se non disponibile, posizionare una bottiglia di vetro in un bicchiere pieno di acqua ghiacciata. A temperature più alte la polossamero sarà superiore al punto di gel e non sciogliere correttamente. <ol><li> Aggiungere 60 ml di soluzione di PBS freddo ghiaccio in una bottiglia di vetro e agitare energicamente con un agitatore magnetico. </li><li> Pesare 24,5 grammi di polossamero e aggiungerlo in piccole quantità per la PBS freddo. Attendere che il polossamero ha parzialmente sciolto prima di aggiungere di più. </li><li> Agitare la soluzione fino a quando tutti polossamero è sciolta. </li><li> Aggiungere PBS freddo fino a raggiungere un volume finale di 100 ml. La concentrazione finale sarà 24,5% w / v </li><li> Smettere di mescolare la soluzione e lasciare riposare a 4 ° C fino a quando bolle e schiuma nella soluzione sono scomparsi. Bolle che sono intrappolati all&#39;interno del gel saranno trasferiti al pcartuccia rinter e porterà a difetti di stampi sacrificali stampati. </li><li> Filtro (filtro 0,22 micron) la soluzione direttamente nella cartuccia di stampa per rimuovere eventuali particelle indesiderate che potrebbero intasare l&#39;ago. La fase di filtraggio deve essere eseguita in una camera fredda (o se non disponibile con punte raffreddati filtro, ecc) per evitare gelificazione del polossalene nel filtro. Mantenere la cartuccia caricata a 4 ° C fino al 30 min prima dell&#39;esperimento. </li></ol> 2. Preparazione della stampante 3D La stampante 3D utilizzata in questo lavoro è stata la &quot;biofabbrica&quot; ​​dal regenHU. La parte estrusione del sistema costituito da più parti. Una cartuccia in pressione nella parte superiore è collegata a un connettore tramite un adattatore luer-lock. Il connettore colma gli spazi tra l&#39;uscita della cartuccia e l&#39;ingresso di una elettrovalvola. All&#39;uscita dell&#39;elettrovalvola, possono essere usati aghi con diametri diversi. Il materiale viene estruso su un substrare che ha luogo ad una fase in movimento mediante aspirazione. Le parti principali del sistema sono descritte nella Figura 1. Altri sistemi basati estrusione possono essere utilizzati per il processo di stampa, e il processo di ottimizzazione deve essere fatto per ogni sistema. <ol><li> Posizionare la valvola a solenoide (diametro ugello 0,3 mm) e l&#39;ago (diametro interno 0,15 millimetri) in distinte da 1,5 ml provette riempiti con acqua ultrapura e metterli in un bagno ad ultrasuoni riscaldato da pulire per 30 min. Sciacquare le valvole puliti con etanolo e asciugare con una pistola di azoto. </li><li> Installare la valvola e l&#39;ago nella stampante e un, pulito cartuccia vuota. </li><li> Applicare 3 bar di pressione al sistema e soffiare residui liquidi dalla valvola installato e ago con aria compressa. Per piccoli diametri ago, è consigliabile avere un filtro (filtro a siringa comune, 0,45 micron dimensione dei pori) installato all&#39;uscita dell&#39;aria compressa per evitare l&#39;entrata di piccole particelle che potrebbero intasare l&#39;ago. </li&gt; <li> Girare la pressione e installare la cartuccia caricata con il polossamero. La cartuccia deve essere tolta dal frigorifero circa 30 minuti prima di montare la cartuccia in modo che il polossamero possa raggiungere temperatura ambiente e gel. </li><li> Applicare pressione di 3 bar al sistema ed erogare polossamero fino a raggiungere la punta dell&#39;ago e viene estruso in un filamento continuo. </li></ol> 3. Ottimizzazione dei parametri di stampa Creare strutture 3D precisi, il processo di stampa deve essere ottimizzato per il materiale e la concentrazione scelta. Seconda della viscosità e il sistema di stampa 3D ogni materiale produrrà un volume di erogazione specifica e spessore linea per un insieme fisso di parametri. <ol><li> Con un software CAD adeguato (in grado di creare file ISO dai disegni), tracciare una riga circa la stessa lunghezza della struttura che si intende stampare. </li><li> Posizionare un microscopio vetrino 25 millimetri x 75 mm x 1mm o qualsiasi altro substrato nella stampante e fissarlo ruotando sul vuoto. </li><li> Nel software della stampante, impostare l&#39;elettrovalvola ad un&#39;elevata frequenza di 50 Hz e impostare una elevata pressione di 3 bar. </li><li> Stampare uno strato di un&#39;unica linea con una velocità di fase di 300 mm / min. </li><li> Ridurre la pressione fino a raggiungere la larghezza linea desiderata. È inoltre possibile controllare il volume che viene estruso attraverso il tempo di apertura della valvola. </li><li> Ridurre la frequenza della valvola fino nessuna linea continua può essere stampato più. Scegliere una frequenza di sopra di questo valore. </li></ol> Nota: Una volta che la larghezza della linea desiderata e linee continue sono raggiunti, determinare la velocità di fase ottimale e spessore di strato cioè il sollevamento dell&#39;ago dopo che uno strato stampato. <ol start="7"><li> Stampare più strati su uno sopra l&#39;altro e vedere se l&#39;ago è in giusta posizione sopra dello strato precedente dopo diversi strati stampati. Regolare lo spessore dello strato (ago ascensore) In modo che ogni strato è stampato sopra quello successivo (Figura 3). </li><li> Diminuire la velocità di fase della tappa da 300 mm / min graduale in modo che gli strati estrusi iniziano e terminano alle stesse posizioni come i precedenti (Figura 4). Velocità troppo elevate causano palco sul palco per essere in movimento prima che il materiale estruso ha toccato il livello precedente. </li><li> Per la stampa delle strutture pilastro seguono passi 3.1.-3.8., Ma invece di disegnare una sola linea disegnare un singolo punto. I parametri da fuoco quando la stampa dei pilastri sono la pressione (regola spessore e diametro pilastro di poloxamer), il tempo di apertura della valvola (volume estruso) e il tempo di residenza della testina di stampa nella posizione in cui il pilastro deve essere depositato . </li><li> Quando i parametri sono ottimizzati, stampa diversi strati di una linea dovrebbe risultare in una parete solida, o in caso di punti, un pilastro. Salvare i parametri per un uso successivo. </li></ol> Utilizzare i parametri rilevati durante la procedura di ottimizzazione da questo punto in poi. <ol><li> Stampare la struttura interna (qui si tratta di un array di pilastro) su un vetrino da microscopio e lasciare asciugare durante la notte. Questo a) riduce la dimensione e lo spessore delle strutture e b) fornisce una migliore aderenza tra la struttura ed il substrato, così lift-off durante il riempimento può essere evitato. </li><li> Con il software CAD, disegnare una struttura che consiste di una parete esterna che circonda la struttura che si intende aver eluito via e riempito. Stampare la struttura con poloxamer. La stampa della parete prenderà 6 min. </li></ol> Attenzione: La parete deve essere stampato almeno 3,5 mm dalla struttura interna a causa delle dimensioni del dell&#39;ago. Altrimenti la stampa della parete esterna distruggerà la struttura interna <ol start="3"><li> Preparare la soluzione che si desidera per riempire il vostro sacrificial con stampo (qui di agarosio 1% in acqua deionizzata). La soluzione di agarosio deve avere una temperatura compresa tra 35 ° C e 45 ° C. Sotto questa temperatura, l&#39;agarosio solidificherà troppo velocemente; sopra di questa temperatura, potrebbe distruggere i pilastri stampati perché la struttura polossamero ammorbidirà. </li><li> Lentamente riempire lo stampo con la soluzione sacrificale riempimento mediante pipetta. Questo dovrebbe essere fatto lentamente per evitare la distruzione della struttura all&#39;interno della parete. </li><li> Lasciate che il gel soluzione ripiena o reticolarlo seconda del polimero utilizzato. Nel caso di agarosio della solidificazione avvenuta a 4 ° C per 10 min. </li><li> Mettere lo stampo sacrificale ripiena in un bagno di ghiaccio per 10 minuti per eluire la struttura poloxamer. </li><li> Asciugare la struttura ripiena con un fazzoletto di carta e metterlo su un nuovo vetrino da microscopio. Premere attentamente la struttura sul vetrino per microscopio per evitare perdite del terzo idrogel dal vuoto nello spazio trala struttura ripiena e il vetrino per microscopio. </li></ol> 5. Riempimento dei vuoti <ol><li> Per riempire i vuoti lasciati dal poloxamer eluito, riempire la soluzione di polimero prevista in una siringa dotata di un ago G 30. In questo esempio, abbiamo usato un alginato metacrilato 1% in soluzione di NaCl 0,15 M con l&#39;aggiunta di 0,05% w / v litio fenil-2 ,4,6-trimethylbenzoylphosphinate (LAP) e 2,5% v / v di Alexa-488 fibrinogeno coniugato . La Alexa-488 fibrinogeno coniugato è stato aggiunto per scopi di visualizzazione. </li><li> Photopolymerize il polimero con una lampada UV ad alta intensità (100 Watt, 365 nm, la distanza dal substrato è stata del 3,5 cm) per 5 minuti e il costrutto immagine usando un microscopio a fluorescenza o confocale. </li></ol>

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Tissue. 6, 302-302 (2012).</li><li>Butterworth, A., Garcia, M. D. L., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photopolymerized+poly(ethylene)+glycol+diacrylate+(PEGDA)+microfluidic+devices.">Photopolymerized poly(ethylene) glycol diacrylate (PEGDA) microfluidic devices.</a> Roy. Soc. Ch. , 4-6 (2005).</li><li>Shachar, M., Tsur-Gang, O., Dvir, T., Leor, J., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+immobilized+RGD+peptide+in+alginate+scaffolds+on+cardiac+tissue+engineering.">The effect of immobilized RGD peptide in alginate scaffolds on cardiac tissue engineering.</a> Acta biomaterialia. 7, 152-162 (2011).</li><li>Jeon, O., Bouhadir, K. H., Mansour, J. M., Alsberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocrosslinked+alginate+hydrogels+with+tunable+biodegradation+rates+and+mechanical+properties.">Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties.</a> Biomaterials. 30, 2724-2734 (2009).</li><li>Mauck, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering+of+articular+cartilage+through+dynamic+loading+of+chondrocyte-seeded+agarose+gels.">Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.</a> J. Biomech. Eng-T Asme. 122, 252-260 (2000).</li><li>D'Arrigo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+methacrylate+derivatives+and+calcium+alginate+interpenetrated+hydrogel+networks+for+biomedical+applications:+physico-chemical+characterization+and+protein+release.">Hyaluronic acid methacrylate derivatives and calcium alginate interpenetrated hydrogel networks for biomedical applications: physico-chemical characterization and protein release.</a> Colloid Polym. Sci. 290, 1575-1582 (2012).</li><li>Pescosolido, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+Acid+and+Dextran-Based+Semi-IPN+Hydrogels+as+Biomaterials+for+Bioprinting.">Hyaluronic Acid and Dextran-Based Semi-IPN Hydrogels as Biomaterials for Bioprinting.</a> Biomacromolecules. 12, 1831-1838 (2011).</li><li>Guo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+of+collagen/chondroitin+sulfate/hyaluronan+interpenetrating+polymer+network+for+cartilage+tissue+engineering.">Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering.</a> J. Mater. Sci-Mater. M. 23, 2267-2279 (2012).</li></ol>

Gli autori non hanno nulla da dichiarare.

Presentiamo qui, per la prima volta, l&#39;uso di un polimero termosensibile per uno stampo sacrificale che può essere rapidamente eluita in acqua fredda a causa del gel-sol transizione di poloxamer di ~ 20 ° C. La velocità dell&#39;intero processo rende polossamero interessante per la rapida creazione di strutture biopolimero che non può essere stampato con una risoluzione adeguata. La tecnica qui descritta può essere utilizzata per patterning un idrogel idrogel all&#39;interno di un altro o per la creazione di ca...

Approcci di ingegneria tissutale hanno fatto molti progressi negli ultimi anni per quanto riguarda la rigenerazione di tessuti e organi umani 19,20. Tuttavia, finora, l&#39;attenzione di ingegneria tissutale è stata spesso limitata ai tessuti che hanno una struttura semplice o piccole dimensioni come la vescica 21,22 o la pelle 23-25. Il corpo umano, tuttavia, contiene molti complessi tessuti tridimensionali in cui le cellule e matrice extracellulare sono disposte in modo spazialmente definito. Di produrre questi tessuti, è richiesta una tecnica che può collocare cellule e matrice extracellulare ponteggi all&#39;interno di un costrutto tridimensionale in posizioni specificate. Bioprinting ha il potenziale di essere un tale tecnica in cui la visione di produzione complessi tessuti tridimensionali può essere realizzato 10,11,26-28. Bioprinting è definito come &quot;l&#39;uso di processi di trasferimento di materiale per patterning e montaggio biologicamente relmateriali nenti - molecole, cellule, tessuti e di biomateriali biodegradabili. - con una organizzazione prescritto di realizzare una o più funzioni biologiche &quot;4 Si comprende diverse tecniche che funzionano a diverse risoluzioni e le scale di lunghezza, che vanno dalla risoluzione sub-micron di due -fotone polimerizzazione 29 ad una risoluzione di 150 micron a 420 micron per la stampa estrusione 1,12,30. Non un unico materiale o combinazione di materiali in grado di soddisfare le esigenze di ciascun metodo 31. Per la stampa di estrusione, i parametri chiave sono viscosità e tempo di gelificazione 32, dove l&#39;alta viscosità e rapido gelificazione sono desiderabili. Stampa 3D è una tecnica che permette la facile creazione di stampi sacrificali per la creazione di geometrie complesse 30,33,34. Questo processo si basa sulla realizzazione di uno stampo utilizzando una tecnica di prototipazione rapida, come un bioprinter estrusione. Lo stampo sacrificale creata viene utilizzataper formare strutture complesse da materiali che sono difficili da stampare a causa della loro bassa viscosità e tempo di gelificazione lento. Il metodo qui presentato prevede la realizzazione di uno stampo sacrificale costituito da un materiale che si dissolve rapidamente a bassa temperatura e può essere estrusa con precisione. Il copolimero a blocchi poli (etilene glicole) 99-poli (propilene glicole) 67-poli (glicole etilenico) 99 (noto anche come Pluronic F127 o poloxamer 407) soddisfa questi requisiti. Esso è già stato usato in una versione modificata nella stampa estrusione 1 ma, a nostra conoscenza, non è mai stata utilizzata per la stampa nella sua versione non modificata a causa della sua instabilità in ambienti liquidi. Polossamero 407 mostra anche un inverso termico comportamento reattivo 18, vale a dire che cambia da un gel ad un sol sul raffreddamento. La cosa più importante, può essere stampato in complesse strutture arbitrariamente curve ad altissima fedeltà. Ciò permette la creazione di un idrogel strutturata da unamateriale a bassa viscosità, in questo caso lento gelificazione agarosio, pipettando la soluzione nello stampo sacrificale stampato. La combinazione di stampa stampo sacrificale con alta fedeltà e l&#39;eluizione rapida dall&#39;idrogel strutturato colato rende un metodo veloce e flessibile per creare stampi con geometrie diverse, senza l&#39;uso di una maschera o un timbro come è spesso richiesto in metodi litografici. L&#39;idrogel strutturato colato può essere ulteriormente riempito con un altro materiale che non è adatto per la stampa estrusione a causa della sua bassa viscosità. Questo è nel nostro caso una soluzione di alginato metacrilato bassa viscosità. Qui vi presentiamo il metodo di termosensibile stampi sacrificali inversa per idrogel patterning con l&#39;esempio di una matrice colonna.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Poloxamer (Pluronic F127)</td>
 <td>Sigma</td>
 <td>P2443</td>
 <td ></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=10010015" target="_blank">10010-015</a></td>
 <td ></td>
 </tr>
 <tr>
 <td>CAD software</td>
 <td>regenHU</td>
 <td>BioCAD</td>
 <td ></td>
 </tr>
 <tr>
 <td>Alginate methacrylate</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td>
 <td>Invitrogen</td>
 <td>F13191</td>
 <td ></td>
 </tr>
 <tr>
 <td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Lonza</td>
 <td>50004</td>
 <td ></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Bioprinter</td>
 <td>regenHU</td>
 <td>Biofactory</td>
 <td ></td>
 </tr>
 <tr>
 <td>Valve</td>
 <td>regenHU</td>
 <td>300 &mu;m Nozzel Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>regenHU</td>
 <td>150 &mu;m Inner Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Zeiss Axioobserver with ApoTome</td>
 <td>Zeiss</td>
 <td></td>
 <td ></td>
 </tr>
 <tr>
 <td>UV Light Source</td>
 <td>UVP</td>
 <td>Blak-Ray B-100AP High Intensity UV Lamp</td>
 <td>100 W</td>
 </tr>
 </tbody>
</table>

printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3d cell culture

Bioprinting è una tecnologia emergente che ha le sue origini nel settore della prototipazione rapida. I diversi processi di stampa possono essere suddivisi in contatto bioprinting 1-4 (estrusione, dip penna e litografia soft), contactless bioprinting 5-7 (transfer laser in avanti, stampa a getto di deposizione) e tecniche basate laser come due fotoni fotopolimerizzazione 8. Può essere usato per molte applicazioni come ingegneria dei tessuti 9-13, 14-16 microfabbricazione biosensore e come strumento per rispondere a domande biologiche di base come influenze di co-coltura di tipi cellulari 17. A differenza dei metodi fotolitografiche o soft-litografica comuni, estrusione bioprinting ha il vantaggio che non richiede una maschera o timbro separato. Utilizzando il software CAD, la progettazione della struttura può essere rapidamente modificato e adeguato in funzione delle esigenze dell&#39;operatore. Questo rende più flessibile che bioprinting litografia basataapprocci. Qui mostriamo la stampa di uno stampo sacrificale per creare una struttura multi-materiale 3D utilizzando una matrice di colonne all&#39;interno di un idrogel come esempio. Questi pilastri potrebbero rappresentare strutture cave per una rete vascolare o dei tubi all&#39;interno di un condotto guida nervosa. Il materiale scelto per lo stampo sacrificale era polossamero 407, un polimero termosensibile con eccellenti proprietà di stampa che è liquido a 4 ° C e un solido sopra della sua temperatura di gelificazione ~ 20 ° C per 24,5% w / v soluzioni 18. Questa proprietà permette lo stampo sacrificale polossamero basata da eluito su richiesta e presenta vantaggi rispetto alla lenta dissoluzione di un materiale solido soprattutto per geometrie strette. Poloxamer è stato stampato su vetrini da microscopio per creare lo stampo sacrificale. Agarosio è stato pipettato nello stampo e raffreddato fino a gelificazione. Dopo l&#39;eluizione del poloxamer in acqua ghiacciata, i vuoti nello stampo agarosio sono stati riempiti con alginato metacrilato spIked con FITC fibrinogeno marcato. I vuoti riempiti sono stati poi reticolato con raggi UV e il costrutto è stato ripreso con un microscopio a fluorescenza.

I risultati rappresentativi mostrano che la tecnica dello stampo inverso (illustrato nella figura 2) creerà un gel strutturato che può essere riempito con un secondo materiale. All&#39;inizio di ogni processo di stampa, i parametri di stampa sono prima ottimizzati. Regolazioni fasi dell&#39;attuale parametri provocheranno costrutti stampati multistrato rappresentati in Figura 3 e Figura 4 quando singole linee vengono stampate. Se lo spessore dello strato (l&#39;ascens...

Watch this Scientific Journal Video about Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture at JoVE.com

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Un bioprinter stato usato per creare idrogel patterned basato su uno stampo sacrificale. Lo stampo poloxamer stata riempita con un secondo idrogel e quindi eluita, lasciare vuoti che sono stati riempiti con una terza idrogel. Questo metodo utilizza eluizione veloce e buona stampabilità di polossalene di generare architetture complesse di biopolimeri.

La stampa termosensibile Stampi inversa per la Creazione di Patterned idrogeli a due componenti per coltura cellulare 3D

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

printing-thermoresponsive-reverse-molds-for-creation-patterned-two

Research

JoVE Journal

Immunology and Infection

15.0K Views.  Cartilage Engineering & Regeneration. Un bioprinter stato usato per creare idrogel patterned basato su uno stampo sacrificale. Lo stampo poloxamer stata riempita con un secondo idrogel e quindi eluita, lasciare vuoti che sono stati riempiti con una terza idrogel. Questo metodo utilizza eluizione veloce e buona stampabilità di polossalene di generare architetture complesse di biopolimeri.

Video: Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Il rilevamento del virus infettivo da Campo-zanzare raccolte mediante test coltura cellulare Vero

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Ricerca

Immunologia e infezioni

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

A primaria del sistema cultura Neuron per lo Studio della latenza del virus Herpes Simplex e la riattivazione

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoraggio migrazione delle cellule dendritiche usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Organoidi come modello per le malattie infettive: cultura dei diritti dell&#39;uomo e organoidi stomaco murini e microiniezione dell&#39;Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Citometria a flusso basato su test per il monitoraggio delle funzioni delle cellule NK

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

Mouse e umano-derivato cultura dello strato monomolecolare epiteliale gastrica primaria per lo studio della rigenerazione

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

Coltura di cellule batteriche a livello di singola cellula all'interno di vescicoli giganti

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

Analisi di cocultura ad alta produttività per l'analisi delle interazioni microbiche

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

Analisi dell'amplificazione isotermica mediata dal ciclo di trascrizione inversa (RT-LAMP) Per il rilevamento specifico e rapido del virus del lago Tilapia

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. aeruginosa Co-cultura 3D infetta di cellule epiteliali bronchiali e macrofagi all'interfaccia aria-liquido per la valutazione preclinica degli anti-infettivi

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...