Gli autori desiderano ringraziare Lisa Dailey per gli ingressi voti. Questo lavoro è stato sostenuto da sovvenzioni dal National Institutes of Health (ES013611 e HL095163), l&#39;assistente di volo Medical Research Institute (FAMRI), e l&#39;Environmental Protection Agency (CR83346301) e dichiara alcun conflitto di interessi. Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del National Institutes of Health. Anche se la ricerca descritta in questo articolo è stato finanziato in parte dalla US Environmental Protection Agency attraverso accordo di cooperazione CR83346301 con il Centro di Medicina Ambientale, Asma e Polmone di Biologia presso la University of North Carolina-Chapel Hill, non è stato sottoposto alla L&#39;agenzia del peer richiesto e revisione della politica e quindi non riflettono necessariamente il punto di vista dell&#39;agenzia, e nessuna approvazione ufficiale dovrebbe essere dedotto. Menzione of nomi commerciali o di prodotti commerciali non costituisce approvazione o raccomandazione per l&#39;uso. Loretta Müller è sostenuta dalla National Science Foundation svizzera con un contributo personale.

1. Preparare Plastic Ware: Cappotto Piatto <ol><li> Aggiungere 500 microlitri PureCol (1:100 diluito con acqua sterile) a ciascun pozzetto di una piastra 12 pozzetti. </li><li> Incubare a 37 ° C in un incubatore per 30 minuti, rimuovere il PureCol e risciacquare con HBSS destra prima che le cellule vengono aggiunti alla piastra (vedere il punto 3.1). </li></ol> 2. Ottenere e pretrattamento Biopsia del CNE umano <ol><li> Nasale biopsia raschiare <ol><li> Ottenere EC superficiali che rivestono i turbinati nasali sotto visione diretta attraverso un speculum riutilizzabili in polipropilene nasale 9 mm (modello 22009) su un otoscopio operativo con speculum (modello 21700). Questo dispositivo fornisce una visualizzazione ottimale dei turbinati nasali e la flessibilità di movimento. </li><li> Eseguire le biopsie con il soggetto seduto in posizione verticale su una sedia a schienale diritto con la testa leggermente piegata all&#39;indietro o sdraiati in posizione supina su un lettino. </li><li> Inserire lo speculum otoscopio nella narice e Turbi inferiorenate visualizzato con illuminazione. </li><li> Inserire una curetta termoplastico sterile attraverso lo speculum con la punta estesa distalmente alla parte posteriore del turbinato. </li><li> Utilizzando una leggera pressione sulla superficie inferiore del turbinato, la curetta viene tracciata attraverso il 5x superficie mucosa e retratto. Un recupero di successo di cellule della mucosa detenute da un&#39;azione capillare sarà evidente nella tazza del curetta. </li></ol></li><li> Posizionare campione in una provetta conica da 15 ml contenente 8 ml di RPMI 1640, trasporto su ghiaccio. </li><li> Usare pinze per recuperare la sonda, rimuovere il tessuto da sonda rinoceronte con una pipetta P-1000, scartare sonda </li><li> Centrifuga a pellet (4 ° C, 400 xg, 5 min), aspirare il surnatante (attenzione: attenzione a non aspirare il pellet). </li><li> Risospendere il pellet in 1 ml BEGM con 10 ml di 100x DNasi 1. </li><li> Incubare a temperatura ambiente per 20 min. </li><li> Centrifuga (4 ° C, 400 xg, 5 min), NON aspirare il surnatante! </li></ol> 3. SEED celle di plastica (giorno 0) <ol><li> Rimuovere il PureCol dalla piastra e risciacquare con HBSS (vedi anche passo 1.2). </li><li> Aggiungere un volume minimo di BEGM supporti + + (80-100 microlitri, fino a visualizzare un menisco di supporto nel centro del pozzetto) in 1-4 pozzetti (a seconda delle dimensioni biopsia) della piastra rivestita. </li><li> Utilizzare una pipetta P-1000 per rimuovere con attenzione il pellet del tessuto bioptico dal tubo, senza aspirazione troppo media. </li><li> Trasferire il pellet in mezzo pozzi, tenere la biopsia insieme come un ammasso di cellule, e non rompere a fare una sospensione singola cella. Un buon rendimento biopsia fino a 4 pozzi che possono essere seminati. Mettere la piastra nel tessuto culturale incubatore (37 ° C, 5% CO 2,&gt; 90% di umidità relativa). </li><li> Controllare dopo due ore per assicurare che vi sia sufficiente supporti (senza disturbare il menisco). </li><li> Day1, 24 ore di post-semina: raccogliere tutte le cellule non aderenti di tutti i pozzetti (piastre tilt e raccogliere mezzi di comunicazione con le cellule in un P-1000, collect tutti i pozzetti in una provetta da centrifuga da 1,5 ml). </li><li> Centrifugare (4 ° C, 400 xg, 5 min). </li><li> Mentre centrifugazione sospensione cellulare: aggiungere circa 250 ml di BEGM + + e 250 microlitri della miscela Mezzi BEGM + alle cellule seminate al giorno 0. </li><li> Ripetere i passaggi 3.1-3.5 per le cellule non aderenti. </li><li> Giorno 2-5: variazione media dei cellule giornalieri, aggiungere 500 mezzi microlitri di ogni bene, ridurre la quantità di BEGM + + supporti graduale (giorno 2 dopo la semina: 125 ml BEGM + + oltre 375 microlitri BEGM +, day3 dopo la semina e nei seguenti giorni: 73 pl BEGM + + più 427 ml BEGM +). </li></ol> 4. Ampliare le cellule nelle Boccette <ol><li> Monitorare le cellule quotidiana; una volta che hanno raggiunto il 80-90% di confluenza (solitamente 5-7 giorni dopo la biopsia, se prendono più essi non crescere con successo) le cellule di sollevamento come segue: i media accuratamente aspirato, aggiungere 500 ml di 0,25% tripsina per pozzetto, tenerli a 37 ° C finché le cellule vengono staccate (richiede solitamente 2-3 min). </li><li> Preparare la necessitàEd volume di soia inibitore della tripsina (SBTI) (750 pl per 1 ml di tripsina) in una provetta da 15 ml. </li><li> Staccare le cellule da pipettare ripetutamente su e giù per la soluzione di tripsina; trasferimento di cellule staccate nel tubo da 15 ml con SBTI. </li><li> Lavare tutti i pozzetti con un totale di 1 ml HBSS, aggiungere HBSS al tubo con le cellule. </li><li> Centrifuga a pellet (4 ° C, 400 xg, 5 min), aspirare i media, risospendere in 1 ml BEGM + supporti e agitare la provetta. </li><li> Espandere le cellule in un pallone T25 o T75 a seconda delle dimensioni del pellet (questo è p1; circa due pozzetti confluenti andare in un pallone T75, uno confluenti bene è abbastanza per un T25, di solito due pozzetti confluenti producono un T75). <ol><li> Aggiungi BEGM + ai palloni (5 ml ciascuno per un T75, 3 ml ciascuno per un T25). </li><li> Aggiungere la sospensione cellulare nei flaconi. Trasferire i flaconi in un tessuto culturale incubatore. </li></ol></li><li> 24 ore post-pallone-semina: aggiungere ulteriori supporti BEGM + al pallone (3 ml ad un T25, 10 ml ad un T75). </li><li><strong&gt; mantenere le cellule nelle boccette: Rimuovere i supporti (aspirato da un angolo) e aggiungere BEGM + supporto (5 ml/T25, 20 ml/T75). Il supporto deve essere cambiato ogni 2-3 giorni. Mantenere le cellule nel pallone fino a circa 80% confluenti (confluenza può non essere ancora tutta la beuta; normalmente questo avviene tra 7-10 giorni, se non sono confluenti dopo 10 giorni non cresceranno correttamente). </li></ol> 5. Seed Celle sul tessuto Cultura Inserti <ol><li> Rimuovere il supporto dal pallone e aggiungere tripsina (3 ml per T25, 4 ml per pallone T75). </li><li> Incubare a 37 ° C finché le cellule vengono staccate (circa 2-3 min). </li><li> Preparare SBTI (750 pl per 1 ml di tripsina&gt; 2,25 ml per T25, T75 per 3 ml) in una provetta da 15 ml. </li><li> Staccare le cellule con nastro adesivo il pallone e pipettando su e giù e trasferimento a tubo conico con il SBTI. </li><li> Lavare il pallone con HBSS (1 ml per T25, 2 ml per T75), aggiungere la HBSS al tubo 15 ml. </li><li> Centrifugaa pellet (4 ° C, 400 xg, 5 min), rimuovere con attenzione il surnatante. </li><li> Preparare i piatti: decidere quanti piatti stanno per essere testa di serie, etichettare i piatti con il codice di esempio e il numero, il numero di passaggio (cioè p2) e la data. Aggiungere 700 microlitri BEGM + supporto alla camera basale. </li><li> Risospendere il pellet di cellule in 1 ml di mezzi BEGM ALI vortex la provetta. </li><li> Contare le cellule con un emocitometro (un T25 solito produce circa 4 milioni di cellule, un T75 può avere fino a 20 milioni di cellule a seconda delle isolotto; utilizzare 1-2 milioni di cellule per una piastra 12 pozzetti) e diluire la sospensione cellulare con supporti BEGM ALI al volume totale necessario per la quantità di piatti che dovrebbe essere testa di serie (1,2 x 10 6 cellule in 2,5 ml di supporti per piastra). (Nota: Se parte delle cellule vogliono essere congelati, etichettare il tubo e rimuovere le cellule qui: di solito congelare 2 x 10 6 cellule in 2 ml di congelamento media) </li><li> Aggiungere 200 microlitri di sospensione cellulare lato apicale di ciascuna cella Corning 12well patinatainserire (&gt; questo sarà di circa 80,000-150,000 cellule / inserto; 12 millimetri transwells con 0,4 micron pori poliestere (polietilene tereftalato (PET)) inserto a membrana); trasferire nella coltura tissutale incubatore. </li><li> Dopo 24 ore, cambiare il supporto apicale (200 microlitri media espansione). </li><li> Il mantenimento delle colture cellulari: Cambiare il supporto (BEGM ALI media; 700 microlitri basolaterale e 200 apicale pl) ogni altro giorno. Inserti devono essere mantenuti sommerso fino celle sono completamente confluenti (senza fori in monostrato sono visibili). A seconda del numero di cellule questo avviene di solito tra 2-6 giorni, se ci vuole più tempo le cellule saranno molto probabilmente non essere praticabile. </li></ol> 6. Stabilire Air Interface Liquid Una volta che le cellule sono completamente confluenti (Quando le cellule sulle Transwells sono completamente confluenti) <ol><li> Una volta che le cellule sono completamente confluenti sostituire entrambi i media apicale e basolaterale con i media BEGM ALI integrato con 500nm acido retinoico per 48 ore. </li><li> 48 ore dopo aver aggiunto l&#39;acido retinoico completato BEGM ALI supporti: Preparare PneumaCult-ALI Maintenance Medium (le istruzioni del seguente produttore): Calcolare il volume di terreno necessari (per un piatto di solito 8,5 ml per i vani basali); aggiungere per 1 ml PneumaCult Complete Piano Base : 10 microlitri PneumaCult 100x Manutenzione Supplemento 2 ml eparina (di una soluzione madre 2 mg / ml) 5 ml idrocortisone (di una soluzione madre 96 microlitri / ml). </li><li> Rimuovere tutti i supporti e aggiungere 700 microlitri PneumaCult-ALI manutenzione dei media al lato basolaterale soltanto (nessun mezzo sul lato apicale). </li><li> Mantenere le colture cellulari per 4 settimane al ALI: <ol><li> Modificare i mezzi di ogni altro giorno (Lunedi, Mercoledì e Venerdì orario funziona bene per noi). </li><li> Dopo una settimana a ALI, una volta a settimana (mercoledì) la superficie apicale viene risciacquato: aggiungere 200 microlitri HBSS al lato apicale, tenerli per 10 minuti in incubatrice, con attenzione aspirare ilHBSS (usare una pipetta di vetro 9 in allegato alla trappola vuoto nella cabina di sicurezza biologica, utilizzare punte 200 microlitri sulla punta della pipetta di aspirazione fuori HBSS). </li></ol></li></ol> Nota: Cambiare punte tra piastre, così come cambiare le punte quando si passa dalla superficie apicale rispetto basolaterale. Dopo circa 2 settimane al ALI, le cellule iniziano a differenziarsi e appaiono ciglia. Le colture cellulari raggiungono la piena differenziazione dopo circa 4 settimane al ALI.

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C., Jaspers, I., Dailey, L., Diaz-Sanchez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+microRNA+expression+in+human+airway+cells+by+diesel+exhaust+particles+is+linked+to+tumorigenesis-associated+pathways.">Disruption of microRNA expression in human airway cells by diesel exhaust particles is linked to tumorigenesis-associated pathways.</a> Environ. Health Perspect. 117, 1745-1751 (2009).</li><li>Horvath, K. M., Brighton, L. E., Herbst, M., Noah, T. L., Jaspers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Attenuated+Influenza+Virus+(LAIV)+induces+different+mucosal+T+cell+function+in+nonsmokers+and+smokers.">Live Attenuated Influenza Virus (LAIV) induces different mucosal T cell function in nonsmokers and smokers.</a> Clin. Immunol. 142, 232-236 (2012).</li><li>Horvath, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+lavage+natural+killer+cell+function+is+suppressed+in+smokers+after+live+attenuated+influenza+virus.">Nasal lavage natural killer cell function is suppressed in smokers after live attenuated influenza virus.</a> Respir. Res. 12, 102 (2011).</li><li>Noah, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke+exposure+and+altered+nasal+responses+to+live+attenuated+influenza+virus.">Tobacco smoke exposure and altered nasal responses to live attenuated influenza virus.</a> Environ. Health Perspect. 119, 78-83 (2011).</li></ol>

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EC respiratorie non solo forniscono una barriera fisica per proteggere il corpo umano da stimoli esogeni 20, ma anche agire come un centralino per avviare e regolare le risposte di difesa immunitario respiratorie 1-3 tramite la secrezione di mediatori solubili o interazioni dirette cellula-cellula recettore-ligando. Per studiare ulteriormente il ruolo di EC nella risposta immunitaria, per esaminare differenza di potenziale tra i CE da popolazioni malate, o per studiare gli effetti dei fattori di st...

Obiettivo della tecnica Le cellule epiteliali (ECS) nelle vie aeree sono tra i primi siti ad essere direttamente esposti ai fattori di stress ambientale per via inalatoria, come agenti patogeni, allergeni o inquinanti atmosferici. EC sono più di una barriera strutturale: Agiscono come un centralino per avviare e regolare la risposta immunitaria respiratorio 1-3 tramite il rilascio di mediatori solubili 4-6 e l&#39;espressione dei ligandi / recettori sulla loro surfac 7-9. Mentre linee cellulari immortalizzate possono essere utilizzati come modelli per studiare i CE respiratorio in vitro, non riescono a differenziarsi in popolazioni cellulari eterogenee composte ciliated polarizzata e cellule che producono muco, simile a quello osservato in vivo. Ricapitolando caratteristiche importanti del epitelio respiratorio osservate in vivo, possono essere usate EC vie aeree primarie. Pertanto, il nostro obiettivo era quello di ottimizzare il nostro metodo utilizza nasale EC (CNE) ha ottenuto da volontari umani. CNE sono espansi e coltivate in vitro, ottenendo un modello di coltura cellulare di CNE differenziate che imita molte delle caratteristiche dell&#39;epitelio nasale visto in vivo. Fondamento logico L&#39;utilizzo di modelli in vitro con cellule endoteliali umane primarie mimano situazione in vivo - come descritto in questo studio - dà possibilità uniche per studiare le malattie specifiche differenze di EC, parametri fisiologici associati con l&#39;epitelio delle vie aeree, ovvero l&#39;interazione tra cellule endoteliali e altri tipi di cellule, come cellule dendritiche 10, cellule natural killer 11 o macrofagi. Diversi metodi esistenti utilizzino EC bronchiali, che possono essere ottenute tramite biopsie invasivo spazzola durante broncoscopie, o altrimenti dismesse da tessuto polmonare 12-17. Inoltre, fonti commerciali per ottenere delle vie respiratorie umane colture di cellule epiteliali completamente differenziate esistono (modello EpiAirway da MatTek, Ashland, MA, Cloneticsda Lonza, Basilea, Svizzera, MucilAir da Epithelix Sars, Ginevra Svizzera), dove i ricercatori possono scegliere tra diversi donatori. Tuttavia, a causa del gruppo limitato di cellule epiteliali disponibili in commercio, delimitato o prescritta di parametri, il costo connesso con l&#39;ottenimento commercialmente vie respiratorie umane primarie EC, e l&#39;accesso limitato al tessuto polmonare scartata o tessuto biopsia bronchiale umano appena ottenuto, proibiscono molti ricercatori dalla realizzazione di studi in piena differenziati EC vie respiratorie umane. Pertanto, abbiamo deciso di sviluppare una tecnica che 1) utilizzare CNE umani non invasivo ottenuti, 2) fornire un protocollo di cedimento culture differenziata epitelio delle vie aeree umana, e 3) essere riproducibile nella maggior parte degli ambienti di laboratorio attraverso una infrastruttura esistente coltura di tessuti. Vantaggi rispetto esistenti Techniques / Modelli Ottenere CNE freschi, rispetto al bronchiale o piccole vie aeree EC, è molto meno invasiva, individuales possono essere sottoposte a biopsia più volte con effetti collaterali significativi e superficiali biopsie raschiare nasali possono essere condotti in popolazioni malate che non sarebbero altrimenti beneficiare di broncoscopie connesse alla ricerca. Non invasive biopsie raschiare superficiali per ottenere LNE consentono al ricercatore di impostare specifiche donatore a priori, tra cui l&#39;età, il sesso, lo stato di malattia, ecc in linea con l&#39;obiettivo della ricerca da compiere. Così, quando si progetta studi di ricerca gli investigatori non sono limitati dai tessuti clinicamente disponibili / campioni epiteliali, l&#39;acquisto di costosi modelli di coltura cellulare differenziati da fornitori commerciali, o di progettazione studi broncoscopia invasive. Inoltre, la facilità di ottenere CNE aumenta la capacità di condurre esperimenti in cellule da donatori diversi, che migliora validazione statistica dei risultati osservati. Infine, il naso è uno dei primi siti ad essere esposti a qualsiasi tipo di stress aerodispersi. Così, generando in organotipicovitro sistemi di coltura che mimano all&#39;epitelio nasale sono utili per studiare inalazione di particelle virali, inquinanti, allergeni o gas, che può essere facilmente ottenuto utilizzando questo sistema di coltura cellulare.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Nasal speculum</td>
 <td>Welch Allyn</td>
 <td>Model 22009</td>
 <td>9 mm, reusable polyproylene</td>
 </tr>
 <tr>
 <td>Operating otoscope</td>
 <td>Welch Allyn</td>
 <td>Model 21700</td>
 <td></td>
 </tr>
 <tr>
 <td>Rhino probe cell cuvette</td>
 <td>Arlington Scientific</td>
 <td>SY-96-092</td>
 <td>bend the head of the cuvette a little more</td>
 </tr>
 <tr>
 <td>12-well culture plates</td>
 <td>Corning</td>
 <td>3512</td>
 <td></td>
 </tr>
 <tr>
 <td>Transwell membrane cell culture inserts</td>
 <td>Corning</td>
 <td>3460</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue culture flask </td>
 <td>Corning</td>
 <td>430639 and 430641</td>
 <td>T25 and T75 vented flask</td>
 </tr>
 <tr>
 <td>RPMI 1640 media (with L-Glutamine)</td>
 <td>Gibco</td>
 <td>11875</td>
 <td></td>
 </tr>
 <tr>
 <td>Bronchial/Tracheal Epithelial Cell Growth Medium</td>
 <td>Cell applications</td>
 <td>511-500</td>
 <td></td>
 </tr>
 <tr>
 <td>BEGM Bronchial Epithelial Cell Growth Medium</td>
 <td>Lonza</td>
 <td>CC-3170</td>
 <td>This comes as a kit of one bottle of medium and vials with supplements.</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate 7.5% solution</td>
 <td>Gibco</td>
 <td>25080</td>
 <td>Use as it is.</td>
 </tr>
 <tr>
 <td>NuSerum</td>
 <td>BD Biosciences</td>
 <td>355104</td>
 <td>Use as it is.</td>
 </tr>
 <tr>
 <td>BAMBANKER freezing media</td>
 <td>BAMBANKER</td>
 <td>302-14681</td>
 <td>Use as it is.</td>
 </tr>
 <tr>
 <td>CoolCell </td>
 <td>Biocision</td>
 <td>HTBCS-136</td>
 <td>(CryoPrep alcohol-free cell freezing container)</td>
 </tr>
 <tr>
 <td>PureCol</td>
 <td>AdvancedBioMatrix</td>
 <td>5005-B</td>
 <td>dilute 1:100 with sterile water (should be roughly 0.03 mg/ml)use 500 &mu;l/well of a 12-well plate</td>
 </tr>
 <tr>
 <td>HBSS with Ca2 and Mg2+</td>
 <td>Gibco</td>
 <td>14025</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAse 1</td>
 <td>Sigma</td>
 <td>DN25</td>
 <td>Dissolve at 1.5 mg/ml, which is a 100x solution, add 10 &mu;l to each ml of media</td>
 </tr>
 <tr>
 <td>Trypsin, 0.25%, 1x, with phenol red</td>
 <td>Gibco</td>
 <td>25200-056</td>
 <td>Use at it is</td>
 </tr>
 <tr>
 <td>Soy Bean Trypsin Inhibitor (SBTI)</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td>Use dissolved in HBSS at a concentration of 1 mg/ml</td>
 </tr>
 <tr>
 <td>Retinoic acid</td>
 <td>Sigma</td>
 <td>R7632</td>
 <td>Make it up 100 &mu;M stock solution (see below), dilute to 10 &mu;M with absolute alcohol, take 5 &mu;l of this and add it to 1 ml BEGM media (our working solution). 
 
 All-trans Retinol has a coefficient of extinction of 52,480 in ethanol at 325 nm. Dissolve 25 mg in 50 ml absolute ethanol. Serial dilutions need to be made, dilute 50 ml of stock in 450 ml absolute ethanol, take 50 ml of this, dilute in 450 ml absolute ethanol, take 50 ml of this, dilute in 450 ml absolute ethanol, and again take 50 ml of this, dilute in 450 ml absolute Ethanol, take 100 ml of this and dilute in 900 ml absolute ethanol and read in the spectrophotometer at 325 nm, this number is then multiplied by its' dilution factor, then divided by the extinction coefficient of 52,480, this will give a number in mM. Make a 100 mM stock (store at -20 &deg;C) and from this stock a 10 mM aliquot is made, the daily retinoic acid needed would be 5 ml of this 10 mM aliquot in 995 ml ALI media.
 Example: 325nm reading is 1.15. 1.15 x 10,000 (dilution factor) / 52,480 = 0.219 = 219 mM, make a 100 mM stock from this, in absolute ethanol.</td>
 </tr>
 <tr>
 <td>DMEM high glucose (1x), liquid, with L-glutamine and sodium pyruvate</td>
 <td>Gibco</td>
 <td>11995</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Pituitary Extract</td>
 <td>Gibco</td>
 <td>13028-014</td>
 <td>Used for BEGM ALI media.</td>
 </tr>
 <tr>
 <td>Nystatin</td>
 <td>Sigma</td>
 <td>N4014-50 mg</td>
 <td>Make up a solution of 3.3 mg/ml.</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Fisher Scientific</td>
 <td>BP1600-100</td>
 <td>Make up a solution of 10 mg/ml solution in milliQ water.</td>
 </tr>
 <tr>
 <td>PneumaCult-ALI media</td>
 <td>StemCell</td>
 <td>5001</td>
 <td>This comes as a kit of one bottle of medium and vials with supplements. For details see below.</td>
 </tr>
 <tr>
 <td>Hydrocortisone </td>
 <td>StemCell</td>
 <td>7904</td>
 <td>Dissolve 2.4 mg hydrocortisone in 1 ml ddH2O (used to make the 200x Hydrocortisone stock solution)</td>
 </tr>
 <tr>
 <td>Heparin Sodium Salt in PBS (2 mg/ml)</td>
 <td>StemCell</td>
 <td>7980</td>
 <td>Use at it is. </td>
 </tr>
 <tr>
 <td>Filter flasks (500 ml)</td>
 <td>Corning</td>
 <td>431097</td>
 <td>0.22 &mu;m pore size</td>
 </tr>
 <tr>
 <td>Filter tubes (50 ml)</td>
 <td>Millipore</td>
 <td>SCGP00525</td>
 <td>Steriflip, 0.22 &mu;m pore size</td>
 </tr>
 <tr>
 <td>Pipette tips - ART Barrier Tips</td>
 <td>Molecular Bioproducts</td>
 <td>2139RI, 2069RI, 2179RI</td>
 <td></td>
 </tr>
 <tr>
 <td>Conical tubes, sterile, 15 ml and 50 ml</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>ddH2O, absolute ethanol, ice</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CO2 incubator</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>refrigerated centrifuge</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>biological safety cabinet</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>pipettes (p2, p200, p1000), 9 in glass pipettes</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>gloves</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>lab coat with tight cuffs</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
 <table border="1">
 <tbody>
 <tr>
 <td>Media and solutions used:</td>
 <td>Recipe</td>
 <td>Used for</td>
 </tr>
 <tr>
 <td colspan="3">General remark: always warm the media to room temperature</td>
 </tr>
 <tr>
 <td rowspan="2">BEGM media</td>
 <td><ul><li> 500 ml BEGM Cell Application media with one aliquot of retinoic acid</li></ul> </td>
 <td>(mix them half-half and filter )</td>
 </tr>
 <tr>
 <td><ul><li> 500 ml BEGM Lonza media with one aliquot of single quot supplements </li></ul></td>
 <td><ul><li> digestion of the biopsy</li></ul> </td>
 </tr>
 <tr>
 <td rowspan="3">BEGM+ media</td>
 <td><ul><li> 50 ml BEGM media </li></ul></td>
 <td><ul><li> maintaining cells on plastic in the plates (passage0) </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 10 &mu;l retinoic acid (working solution)</li></ul> </td>
 <td><ul><li> seeding cells in the flask (passage1; partially)</li></ul> </td>
 </tr>
 <tr>
 <td></td>
 <td><ul><li> maintain the cells in the flask </li></ul></td>
 </tr>
 <tr>
 <td rowspan="3">BEGM++ media</td>
 <td><ul><li> 50 ml Lonza's BEGM media with supplements</li></ul> </td>
 <td><ul><li> seeding fresh cells in 12-well plate on plastic</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 1 ml Sodium BiCarb</li></ul> </td>
 <td><ul><li> maintaining cells in the plates on plastic (passage0; only partially)</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 2.5 ml NuSerum</li></ul> </td>
 <td><ul><li> seeding cells in the flask (passage1; partially)</li></ul> </td>
 </tr>
 <tr>
 <td rowspan="5">BEGM ALI media</td>
 <td><ul><li> 250 ml DMEM-H </li></ul></td>
 <td rowspan="5"><ul><li> Maintaining cells on the transwell before they go ALI</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 250 ml BEGM including supplements</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 2 ml Bovine Pituitary Extract (12.5 mg/ml solution)</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 1 ml Nystatin (3.3 mg/ml solution)</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 75 &mu;l BSA (10 mg/ml solution)</li></ul> </td>
 </tr>
 <tr>
 <td rowspan="5">200X Hydrocortisone Stock Solution (96 &mu;l/ml solution)</td>
 <td><ul><li> 200 &mu;l dissolved hydrocortisone</li></ul> </td>
 <td rowspan="5"><ul><li> As a supplement to the PneumaCult-ALI Complete Base Medium for the last day in the flask and when cells are on inserts</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 4250 &mu;l dissolved sodium chloride </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 540 &mu;l ddH2O</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 10 &mu;l absolute ethanol</li></ul> </td>
 </tr>
 <tr>
 <td>Filter solution through 0.22 &mu;m filter, aliquot solution and store at -20 &deg;C, avoid additional freeze/thaw cycles</td>
 </tr>
 <tr>
 <td rowspan="3">PneumaCult-ALI Complete Base Media</td>
 <td><ul><li> 450 ml PneumaCult-ALI Basal Medium</li></ul> </td>
 <td><ul><li> Base medium for all three PneumaCult-ALI media </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 50 ml PneumaCult 10x Supplement </li></ul></td>
 <td><ul><li> The PneumaCult media is light sensitive, thus always keep it in the dark</li></ul> </td>
 </tr>
 <tr>
 <td>(this is stable for 2 weeks at 2-8 &deg;C or for up to 6 months at -20 &deg;C)</td>
 <td></td>
 </tr>
 <tr>
 <td rowspan="4">PneumaCult-ALI Maintenance Medium</td>
 <td>Per 1 ml of PneumaCult-ALI Complete Base Medium add:</td>
 <td rowspan="4">ALI culture on inserts</td>
 </tr>
 <tr>
 <td><ul><li> 10 &mu;l PneumaCult 100x Maintenance Supplement </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 2 &mu;l Heparin (of a 2 mg/ml stock solution) </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 5 &mu;l Hydrocortisone (of a 96 &mu;l/ml stock solution) </li></ul></td>
 </tr>
 </tbody>
</table>
 </td>
 </tr>
 </tbody>
</table>

culturing of human nasal epithelial cells at the air liquid interface

Modelli in vitro utilizzando cellule epiteliali umane primarie sono essenziali per comprendere funzioni chiave epitelio respiratorio nel contesto di infezioni microbiche o agenti inalati. Il confronto diretto di cellule ottenute da popolazioni malate ci permettono di caratterizzare diversi fenotipi e sezionare i meccanismi di fondo che mediano i cambiamenti nella funzione delle cellule epiteliali. Coltura di cellule epiteliali della regione tracheobronchiale umana è stata ben documentata, ma è limitata dalla disponibilità di tessuto polmonare umano o invasività associata a ottenere i pennelli bronchiali biopsie. Cellule epiteliali nasali sono ottenuti attraverso molto meno invasivi superficiali biopsie raschiare nasale e soggetti possono essere sottoposte a biopsia più volte senza effetti collaterali significativi. Inoltre, il naso è il punto di ingresso per il sistema respiratorio e quindi uno dei primi siti ad essere esposti a qualsiasi tipo di stress per via aerea, quali agenti microbici, inquinanti o allergeni. Brevemente, le cellule epiteliali nasali ottenuti da volontari umani sono espansi su piastre di coltura dei tessuti rivestiti, e poi trasferite su inserti di coltura cellulare. Dopo aver raggiunto la confluenza, le cellule continuano ad essere coltivate all&#39;interfaccia aria-liquido (ALI), per diverse settimane, il che crea più fisiologicamente condizioni pertinenti. La condizione di cultura ALI usa mezzi definiti che conducono ad un epitelio differenziato che presenta caratteristiche morfologiche e funzionali simili all&#39;epitelio nasale umana, con le cellule ciliate sia e producono muco. Inserti di coltura tissutale con cellule epiteliali nasali differenziate possono essere manipolati in vari modi a seconda delle domande di ricerca (trattamento con agenti farmacologici, trasduzione con vettori lentivirali, esposizione a gas o infezione da agenti microbici) ed analizzati per numerosi endpoint differenti che vanno dalla vie cellulari e molecolari, ch funzionaleanges, morfologia, ecc Nei modelli in vitro di cellule epiteliali nasali umane differenziati consentirà ai ricercatori di affrontare nuove e importanti domande di ricerca utilizzando modelli sperimentali organotipica che gran parte imitare l&#39;epitelio nasale in vivo.

CNE coltura seguendo il protocollo qui descritto re-differenziarsi in uno strato eterogeneo di EC composto da cellule ciliate e non ciliate, che rappresentano la situazione in vivo (Figura 1). Alcuni dei CNE differenziati sono muco che producono cellule (colorazione in blu con AB / PAS, Figura 1B). Anche se il protocollo qui presentato è ottimizzato, differenziazione può variare in relazione alla distribuzione delle popolazioni cellulari totali (cioè differenti rapp...

Watch this Scientific Journal Video about Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface at JoVE.com

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Cellule epiteliali nasali, ottenuti attraverso superficiale raschiare la biopsia di volontari umani, vengono ampliati e trasferite su inserti di coltura tissutale. Dopo aver raggiunto la confluenza, le cellule sono coltivate a interfaccia liquido aria, ottenendo colture di cellule ciliate e non ciliate. Colture di cellule epiteliali nasali differenziate offrono modelli sperimentali validi per lo studio della mucosa respiratoria.

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

culturing-of-human-nasal-epithelial-cells-at-the-air-liquid-interface

Research

JoVE Journal

Biology

36.6K Views.  The University of North Carolina at Chapel Hill.  Cellule epiteliali nasali, ottenuti attraverso superficiale raschiare la biopsia di volontari umani, vengono ampliati e trasferite su inserti di coltura tissutale. Dopo aver raggiunto la confluenza, le cellule sono coltivate a interfaccia liquido aria, ottenendo colture di cellule ciliate e non ciliate. Colture di cellule epiteliali nasali differenziate offrono modelli sperimentali validi per lo studio della mucosa respiratoria. 

Video: Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Primario cellule epiteliali bronchiali umane Grown da espianti

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Ricerca

Biologia

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Un metodo per la selezione di aptameri-struttura di commutazione Applicato ad una colorimetrico oro nanoparticelle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

Progettazione e sviluppo di aptameri delle nanoparticelle d&#39;oro Based colorimetriche saggi per applicazioni in-the-field

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

L'ingestione di fluorescente, nanoparticelle magnetiche per determinare la capacità di assorbimento di liquido negli insetti

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

Differenza di potenziale nasale per quantificare il trasporto Trans-epiteliale ionico in topi

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

Un modello epiteliale bronchiale interfaccia aria-liquido per un'esposizione realistica e ripetuta all'inalazione alle particelle trasportate dall'aria per i test di tossicità

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Sondare la cinetica dell'mRNA nello spazio e nel tempo in Escherichia coli utilizzando la fluorescenza a singola molecola a due colori in ibridazione situ

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

Isolamento delle cellule epiteliali dal follicolo dentale umano

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...