Gli autori desiderano ringraziare Maurice Smith e Marja Talikka per la loro revisione del manoscritto.

<ol>
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E., Shaykhiev, R., Wang, R., Crystal, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+airway+epithelium+air&#8211;liquid+cultures+represent+the+in+vivo+airway+epithelium+transcriptome.">Do airway epithelium air&#8211;liquid cultures represent the in vivo airway epithelium transcriptome.</a> American journal of respiratory cell and molecular biolog. 44, 465 (2011).</li><li>Wiszniewski, 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=Long-term+cultures+of+polarized+airway+epithelial+cells+from+patients+with+cystic+fibrosis.">Long-term cultures of polarized airway epithelial cells from patients with cystic fibrosis.</a> American Journal of Respiratory Cell and Molecular Biolog. 34, 39-48 (2006).</li><li>Balharry, D., Sexton, K., B&#233;ruB&#233;, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+approach+to+assess+the+toxicity+of+inhaled+tobacco+smoke+components:+nicotine,+cadmium,+formaldehyde+and+urethane.">An in vitro approach to assess the toxicity of inhaled tobacco smoke components: nicotine, cadmium, formaldehyde and urethane.</a> Toxicolog. 244, 66-76 (2008).</li><li>Mathis, C., 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=Human+bronchial+epithelial+cells+exposed+in+vitro+to+cigarette+smoke+at+the+air-liquid+interface+resemble+bronchial+epithelium+from+human+smokers.">Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers.</a> Am J Physiol Lung Cell Mol Physio. 304, L489-L503 (2013).</li><li>Cho, M. -. H., 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=A+bioluminescent+cytotoxicity+assay+for+assessment+of+membrane+integrity+using+a+proteolytic+biomarker.">A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker.</a> Toxicology in Vitr. 22, 1099-1106 (2008).</li><li>Stewart, C. E., Torr, E. E., Mohd Jamili, N. H., Bosquillon, C., Sayers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Differentiated+Human+Bronchial+Epithelial+Cell+Culture+Systems+for+Asthma+Research.">Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research.</a> Journal of Allerg. 2012, 11 (2012).</li><li>Blume, L. F., Denker, M., Gieseler, F., Kunze, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+corrected+transepithelial+electrical+resistance+(TEER)+measurement+to+quantify+rapid+changes+in+paracellular+permeability.">Temperature corrected transepithelial electrical resistance (TEER) measurement to quantify rapid changes in paracellular permeability.</a> Pharmazi. , 19-24 (2010).</li><li>Chhin, B., 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=Ciliary+beating+recovery+in+deficient+human+airway+epithelial+cells+after+lentivirus+ex+vivo+gene+therapy.">Ciliary beating recovery in deficient human airway epithelial cells after lentivirus ex vivo gene therapy.</a> PLoS genetic. 5, e1000422 (2009).</li><li>Jiao, J., Meng, N., Wang, H., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+nasal+epithelial+cells+grown+as+explant+outgrowth+cultures+or+dissociated+tissue+cultures+in+vitro.">Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro.</a> Front Me. 7, 486-491 (2013).</li><li>Talbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assessment+of+reproductive+toxicity+of+tobacco+smoke+and+its+constituents.">In vitro assessment of reproductive toxicity of tobacco smoke and its constituents.</a> Birth defects research. Part C, Embryo today : review. 84, 61-72 (2008).</li><li>Port, J. 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=Tobacco+smoke+induces+CYP1B1+in+the+aerodigestive+tract.">Tobacco smoke induces CYP1B1 in the aerodigestive tract.</a> Carcinogenesi. 25, 2275-2281 (2004).</li><li>Hoeng, J., 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=A+network-based+approach+to+quantifying+the+impact+of+biologically+active+substances.">A network-based approach to quantifying the impact of biologically active substances.</a> Drug Discov Toda. 17, 413-418 (2012).</li><li>Iskandar, A. R., 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=Systems+approaches+evaluating+the+perturbation+of+xenobiotic+metabolism+in+response+to+cigarette+smoke+exposure+in+nasal+and+bronchial+tissues.">Systems approaches evaluating the perturbation of xenobiotic metabolism in response to cigarette smoke exposure in nasal and bronchial tissues.</a> Biomed Res In. 2013, 512086 (2013).</li><li>Karp, P. H., 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=An+in+vitro+model+of+differentiated+human+airway+epithelia.+Methods+for+establishing+primary+cultures.">An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures.</a> Methods in molecular biolog. 188, 115-137 (2002).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ISO 3402: Tobacco and tobacco products -- Atmosphere for conditioning and testing. , (1999).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A Language and Environment for Statistical Computing. , (2011).</li><li>Gautier, L., Moller, M., Friis-Hansen, L., Knudsen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+mapping+of+probes+to+genes+for+Affymetrix+chips.">Alternative mapping of probes to genes for Affymetrix chips.</a> BMC Bioinformatic. 5, 111 (2004).</li><li>Hermida, 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=Confero:+an+integrated+contrast+data+and+gene+set+platform+for+computational+analysis+and+biological+interpretation+of+omics+data.">Confero: an integrated contrast data and gene set platform for computational analysis and biological interpretation of omics data.</a> BMC genomic. 14, 514 (2013).</li><li>Thomson, T. M., 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=Quantitative+assessment+of+biological+impact+using+transcriptomic+data+and+mechanistic+network+models.">Quantitative assessment of biological impact using transcriptomic data and mechanistic network models.</a> Toxicology and applied pharmacolog. 272, 863-878 (2013).</li><li>Teff, Z., Priel, Z., Gheber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+forces+applied+by+cilia+depend+linearly+on+their+frequency+due+to+constant+geometry+of+the+effective+stroke.">The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke.</a> Biophysical journa. 94, 298-305 (2008).</li><li>Hilgendorf, C., 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=Caco-2+versus+Caco-2/HT29-MTX+co-cultured+cell+lines:+permeabilities+via+diffusion,+inside-+and+outside-directed+carrier-mediated+transport.">Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.</a> J Pharm Sc. 89, 63-75 (2000).</li><li>Sutto, Z., Conner, G. 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Questo studio è stato finanziato da Philip Morris International.

Qui, abbiamo dimostrato l&#39;applicabilità bronchiale organotipica umana e modelli tessuti nasali per valutare l&#39;impatto di esposizione ripetuta CS. In alternativa alla sperimentazione animale, un certo numero di sistemi di esposizione sono stati sviluppati per le valutazioni tossicologiche dell&#39;esposizione aerosol in vitro (ad es., Vitrocell, Cultex, ALICE, etc.). Questi moduli di esposizione possono anche essere utilizzati per una valutazione tossicologica di inquinanti nell&#39;aria, particelle sospese nell&#39;aria, nanoparticelle, ecc In questo studio, abbiamo utilizzato il sistema Vitrocell che può ospitare fino a 48 campioni diversi contemporaneamente, consentendo sperimentazioni su scala più ampia e bassa variabilità tra i trattamenti. Per ogni esposizione aerosol in vitro, il rischio di contaminazione coltura tissutale rimane un rischio maggiore il cui mitigazione richiede un accurato trattamento delle colture di tessuto durante gli esperimenti. Misurazione deposito di particelle in tempo reale sul microbilancia durante la eesperimento Xposure permette di monitorare che le dosi CS generati dal sistema di esposizione sono conformi alle aspettative. Per garantire la precisione della deposizione di particelle misurata, messa a punto la misura in linea correttamente prima dell&#39;esposizione è critial, ad esempio, la creazione di scala a 0. Inoltre, a causa della differenza tra le aree del microbilancia (cm 2) e la inserto coltura tissutale (0,33 cm 2), abbiamo regolato il calcolo finale alla zona dell&#39;inserto coltura: solo il 33% della deposizione sul microbilancia riflette la deposizione effettiva nell&#39;inserto cultura. Misurazione di TEER accertare funzionalità barriera a tenuta giunzione e per valutare interruzione dello strato epiteliale è una procedura relativamente facile da implementare, che abbiamo riportato qui. Tuttavia, poiché i modelli di tessuto bronchiali e nasali contengono cellule calice intervallati producono muco, lavaggio apicale deve essere eseguita prima delle meas Teermi-. Il lavaggio apicale è critica perché la presenza dello strato di muco e la variabilità del suo spessore può influenzare la misura della TEER, interferire con l&#39;impatto dell&#39;esposizione CS. Questa nozione è in accordo con quanto riportato da Hilgendorf e colleghi, in cui la permeabilità delle cellule Caco-2 è stata influenzata dalla co-coltura con la linea cellulare calice-muco produzione HT29 23. Il muco ha bisogno di essere lavato via prima della misurazione TEER, perché il lavaggio apicale destra prima dell&#39;esposizione può interferire con le risposte del tessuto a CS. Pertanto, la misura viene eseguita tre giorni prima dell&#39;esposizione, e non appena prima dell&#39;esposizione. Abbiamo dimostrato che l&#39;attività CYP1A1 / CYP1B1 potrebbe essere misurata dai modelli culturali organotipica a seguito di esposizione CS, anche se l&#39;attività è stata solo modesta aumentato del CS. Questo segnale debole può essere amplificato da una incubazione più lungo del substrato CYP1A1 / 1B1 (es., Luciferina-CEE), ad esempio per24 hr (dati non mostrati). Uno dei limiti della misurazione dell&#39;attività CYP nel presente lavoro è l&#39;assenza di normalizzazione al livello proteico CYP o per il conteggio delle cellule, che potrebbe essere preso in considerazione per studi futuri per garantire che l&#39;alterazione sull&#39;attività enzimatica non è influenzata dall&#39;alterazione sia del livello di proteina o la conta delle cellule. Abbiamo segnalato che l&#39;esposizione CS inibito l&#39;ciliare battendo sia nel nasale e modelli di tessuto bronchiale. Osservazioni analoghe sono state fatte in diversi modelli di mammiferi e non mammiferi 12. Per ciliare battendo misurazione, assicurando che i tessuti sono trattati e trattati in modo analogo è critica, per esempio se è implementata medio cambiamento, dovrebbe essere applicata per tutti i campioni. Sutto e colleghi hanno riferito che il pH colpito il ciliare mammiferi battendo frequenza 24. Così, quando si confrontano battendo frequenze tra cellule trattate con diverse compunds / miscele, pHregolazione dovrebbe essere considerata per minimizzare la variabilità della misurazione battito ciliare. Inoltre, la temperatura alla quale la misura viene condotta è anche critica come la frequenza del battito ciliare scende al diminuire della temperatura. Per ridurre al minimo la variabilità a causa di questi cambiamenti, un incubatore fase-top, attrezzate con la temperatura, CO 2, e il controllo dell&#39;umidità è stato utilizzato in questo studio. Nonostante questo, abbiamo osservato che il ciliare battendo frequenze nei tessuti bronchiali dopo l&#39;esposizione CS erano molto variabile (es., Di aumentare in un inserto e diminuire nell&#39;altro inserto), suggerendo che il battito ciliare è giusto altamente disturbato dopo l&#39;esposizione. Al contrario, abbiamo osservato l&#39;assenza di frequenze misurabili battito ciliare nel tessuto nasale dopo esposizione CS, suggerendo che la risposta del tessuto nasale è più sensibile e coerente. Ciò è in accordo con una precedente pubblicazione dimostrano che il tessuto nasale ha una capacità inferiore a disintossicarsi comerispetto bronco 25. Infine, abbiamo dimostrato che l&#39;espressione del gene profiling dal bronchiale organotipica e modelli tessuti nasali esposto al CS potrebbe dimostrare un impatto di CS sul metabolismo xenobiotico. È interessante notare che l&#39;alterazione osservata nel metabolismo xenobiotico nel bronchiale organotipica nasale modelli in vitro esposto a CS assomigliano che la situazione in vivo nei fumatori come discusso in maggior dettaglio in una pubblicazione precedente 15. Per le analisi di espressione genica, utilizzando lo strumento robotico rende possibile l&#39;analisi ad alta produttività. Inoltre, la manipolazione robotizzata automatica aumenta ulteriormente la coerenza e la precisione dei risultati di espressione genica. Tuttavia, veloce raccolta dei campioni di tessuto è stato fondamentale per evitare la degradazione dell&#39;RNA durante l&#39;estrazione di RNA. Il trattamento RNA e approcci trascrittomiche qui descritte possono anche essere applicati a campioni di tessuto in vivo.

Lungs are directly and constantly exposed to inhaled air that may contain diverse toxicants such as pollutants and constituents of cigarette smoke (CS). Studying the impact of exposure to those toxicants on respiratory tissues is most informative when done in a manner that resembles in vivo exposure. Compared with the classical 2D immersed cell cultures (e.g., normal human bronchial epithelial cells (NHBE)), 3D organotypic tissue models better recapitulate the morphological, physiological, and molecular aspects of the human airway epithelium in vivo1,2: the 3D tissue models contain the diversity of the cell types observed in vivo, including differentiated epithelial cells, ciliated and non-ciliated cells, goblet cells, and basal cells. They have functional tight junctions and exhibit a mucociliary phenotype 1-3. Moreover, the cultures can be grown on a permeable porous membrane, in an air-liquid interface, allowing a direct exposure to aerosol at the apical side (whereas the basolateral side is immersed in culture medium) 3-5. Dvorak and colleagues reported that gene expression profiles of bronchial tissue models were similar to those obtained from human bronchial brushings 3. In addition, Mathis and colleagues showed that the responses of these tissue models to CS were similar to the differences observed between bronchial epithelial cells obtained from smokers and cells obtained from non-smokers 6. Finally, because the bronchial tissue models could be cultured for up to several months 4,5, they could potentially be used to examine the effects of long-term exposure of test items. 
Cytotoxicity assessments are common parameters measured following chemical insults or to assess the toxicity of specific compounds or mixtures. For instance, membrane integrity can be measured by a luminescent assay and allows the measurement of a dose-dependent cytotoxic effect on the cell culture 7. However, to assess pathophysiological effects of compounds at subtoxic concentrations, other parameters should be measured. For example, tissue integrity determined using the transepithelial electrical resistance (TEER) assay ensures the functionality of tight junctions and monitors the disruption of the epithelial layer 8,9. Ciliary beating frequency also allows the measurement of CS-related effects on respiratory tissues. A normal beating frequency for the cilia lining bordering the upper and lower respiratory tract is important to protect against airway infections 10. Each of the ciliated columnar epithelial cells of the respiratory epithelium has 200-300 cilia beating at a particular frequency to eliminate infectious agents or inhaled particulate matter trapped in the mucus released by interspersed goblet cells 11. CS contains chemicals that may inhibit ciliary beating 12, leading to a reduced protection of the respiratory tract. This work shows that ciliary beating can be measured in organotypic tissue models. This approach allows assessment of whether epithelial cells exhibit their normal function in the organotypic tissue culture. CS also activates xenobiotic metabolism responses in the respiratory tract to metabolize tobacco smoke constituents 13. The activity of the phase I xenobiotic metabolism enzymes, CYP1A1 and CYP1B1, of the tissue models can be measured. Additionally, as previously reported, global gene expression can be measured in the organotypic bronchial tissue models 6,14,15. A transcriptomic data and network-based systems biology approach is leveraged to assess the impact of CS on xenobiotic metabolism 15. 
The methodologies used to expose organotypic 3D bronchial and nasal tissue models to mainstream CS using an in vitro exposure system and to measure the tissue responses to this exposure compared to fresh air exposure (control) are detailed here.

<table border="0" cellpadding="0" cellspacing="0" width="1545">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Name of Material/ Equipment</td>
			<td style="width:367px;">Company</td>
			<td style="width:655px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MucilAir-human fibroblasts-bronchia</td>
			<td>Epithelix S&#225;rl, Geneva, Switzerland</td>
			<td>http://www.epithelix.com/content/view/122/19/lang,en/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MucilAir Culture Medium</td>
			<td>Epithelix S&#225;rl, Geneva, Switzerland</td>
			<td>http://www.epithelix.com/content/view/84/16/lang,en/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VITROCELL</td>
			<td>VITROCELL systems GmbH, Waldkirch, Germany</td>
			<td>http://www.vitrocell.com/index.php?Nav_Nummer=2&#38;R=</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3R4F reference cigarette</td>
			<td>University of Kentucky</td>
			<td>http://www2.ca.uky.edu/refcig/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30-port carousel smoking machine SM2000</td>
			<td>Philip Morris, Int.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CiliaMetrix camera and software</td>
			<td>La Haute &#201;cole de Gestion (HESGE), Geneva, Switzlerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMIL microscope&#160;</td>
			<td>Leica, Heerbrugg, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED light source</td>
			<td>Titan Tool Supplies, Buffalo, NY</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chopstick Electrode STX-2</td>
			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/stx2-chopstick-electrode-set-for-evom2/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EVOMXTM Epithelial Voltohmmeter&#160;</td>
			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/evom2-evom2-epithelial-voltohmmeter-for-teer/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferin Detection Reagent&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagNA Lyser Instrument&#160;</td>
			<td>Roche</td>
			<td>http://www.roche.com/products/product-details.htm?region=us&#38;type=product&#38;id=66</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chloroform&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&#38;region=</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAcube&#160;</td>
			<td>Qiagen</td>
			<td align="right">9001882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>http://www.nanodrop.com/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agilent 2100 Bioanalyzer&#160;</td>
			<td>Agilent</td>
			<td>http://www.genomics.agilent.com/en/Bioanalyzer-System/2100-Bioanalyzer-Instruments/?cid=AG-PT-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Affymetrix GeneChip High throughput 3&#8217;IVT Express Kit</td>
			<td>Affymetrix</td>
			<td>http://www.affymetrix.com/catalog/prod370001/AFFY/High-Throughput-(HT)-Whole-Transcript-(WT)-Kit</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanner 3000 7G&#160;</td>
			<td>Affymetrix</td>
			<td>http://www.affymetrix.com/catalog/131503/AFFY/Scanner-3000-7G</td>
			<td></td>
		</tr>
	</tbody>
</table>

impact assessment of repeated exposure of organotypic 3d bronchial and nasal tissue culture models to whole cigarette smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

Nei risultati di seguito descritti, i tessuti organotipica erano cellule primarie umane epiteliali isolati da sani, non fumatori, i donatori caucasici e sono state ricostituite usando fibroblasti 15. Bronchiale 3D e modelli di tessuto nasale In bronchiale vitro e modelli organotipica nasali assomigliare a livello cellulare epitelio respiratorio umano (Figura 2). L&#39;esposizione CS Bronchiale Organotipica e modelli tessuti nasali possono essere ripetutamente esposti ad integrare CS all&#39;interfaccia aria-liquido. La procedura sperimentale esposizione al fumo utilizzato per i risultati qui illustrati sono illustrati nella figura 1. Registrazione del battito ciliare Cilia pestaggio è stato registrato nei tessuti esposti ad aria come illustrato nel video. L&#39;esposizione CS è stata associata con una riduzione della frequenza ciliare pestaggio: il picco più forte osservata intorno4 Hz nel sham esposti e prima esposizione non si osserva più dopo l&#39;esposizione CS (Figura 3). Questa frequenza ridotta renderebbe le ciglia battendo meno efficiente per rimuovere muco e agenti infettivi 22. Misura di TEER e CYP1A1 / attività 1B1 TEER è stata misurata 48 ore dopo l&#39;ultima esposizione per assicurare il tessuto è ancora funzionante. CYP1A1 / 1B1 attività è stata anche misurata 48 ore dopo la fine dell&#39;esposizione come illustrato in figura 1. I valori TEER nei tessuti CS-esposti non erano significativamente differenti rispetto ai tessuti-aria esposto conferma che non vi è alcun grave effetto citotossico di fumo a questa concentrazione. Al 48 h post-esposizione, CYP1A1 / 1B1 attività dei tessuti era leggermente aumentato, indicando un modesto aumento di tessuto meccanismo di difesa dopo l&#39;esposizione (Figura 4). Profili di espressione genica e sistemi basati sulla rete biapproccio ology per studiare l&#39;impatto dell&#39;esposizione CS sulla alterazione del metabolismo xenobiotici I tessuti sono stati raccolti in 48 h post-esposizione punti di tempo. Successivamente, l&#39;analisi microarray è stata condotta per generare profili di espressione genica del CS e tessuti-aria esposti. Impatto dell&#39;esposizione CS sul metabolismo xenobiotico è stato studiato con un approccio di biologia dei sistemi network-based sfruttato dai profili di espressione genica e, come riferito in precedenza 15. Usando un approccio di biologia dei sistemi basati sulla rete mostra che CS impatti esposizione a livello dei nodi di backbone nel modello di rete Metabolism Xenobiotic erano ben correlati fra in vitro e in vivo set di dati (Figura 5). Al contrario, a livello di ogni singolo cambiamenti di espressione genica, correlazioni poveri sono stati osservati tra in vitro (CS-esposto vs. aria esposto) e in vivo (fumatori vs fumatori). Figura 1. Procedura schematica della esposizione ripetuta di CS ai modelli di tessuto bronchiale organotipica Abbreviazioni:. CYP, del citocromo P450; CS, fumo di sigaretta; TEER, transepiteliale resistenza elettrica. <img alt="Figura 2" src="/files/ftp_upload/52325/52325fig2.jpg" /> Figura 2. Organotipica bronchiali e nasali modelli di coltura di tessuti epiteliali. Fumetto illustra bronchiali (A) e nasale (B) dell&#39;inserto coltura tissutale all&#39;interfaccia aria-liquido. I modelli in vitro contenevano cellule ciliate mostrati nello strato apicale delle cellule colorate ematossilina e eosina (C per bronchiale, D per nasale) come riportato in precedenza 15. I modelli sono stati co-coltura con fibroblasti che sono importanti<html> per la crescita e la differenziazione delle cellule epiteliali (indicato dalle frecce). La colorazione dei marcatori lignaggio delle vie aeree: p63 (E per bronchiale, F per nasale) e MUC5AC (G per bronchiale, H per nasale) sono indicati come riportato in precedenza 15. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52325/52325fig3.jpg" /> Figura 3. Cilia Vincere Frequency. Cilia frequenza battito (CBF) è stata misurata in aria a vista e nella cultura, prima e dopo l&#39;esposizione. Diminuzione CBF è stato visto dopo l&#39;esposizione CS (risultati rappresentativi di due inserti sono mostrati come replicare 1 (R1) e replicano 2 (R2)). annuncio / 52325 / 52325fig4.jpg &quot;/&gt; Figura 4. Misura del CYP1A1 / attività CYP1B1 e TEER. Pannelli sinistra indicano l&#39;attività di CYP1A1 / CYP1B1 nel bronchiale (A) e nasale (B) modelli di tessuto. Pannelli destra indicano la misura TEER nella bronchiale (C) e nasale (D) modelli di tessuto. Sono mostrati mezzi ± SD. Abbreviazioni: CS, fumo di sigaretta; CYP, citocromo P450; TEER, transepiteliale resistenza elettrica; RLU, unità luminescense relativa. <img alt="Figura 5" src="/files/ftp_upload/52325/52325fig5.jpg" /> Figura 5. sistemi basati Network approccio biologico per la valutazione di impatto dell&#39;esposizione CS nel contesto del metabolismo xenobiotico. I cambiamenti dei livelli di espressione genica sono stati usati per calcolare l&#39;attivazione del nodo di backbone del modello di rete Xenobiotic metabolismo (A) </<html> strong&gt; come riportato in una pubblicazione precedente 15. La figura a destra illustra il concetto di quantificazione dei nodi di backbone, noto anche come &quot;valore backbone di rete differenziale,&quot; utilizzando l&#39;approccio NPA. I ovali blu rappresentano l&#39;attività dei nodi di backbone (ie., Lo strato funzionale) e le sfere verdi rappresentano l&#39;espressione di geni (ie., Lo strato trascrizionale). Impatti di esposizione CS nei tessuti bronchiali organotipiche (in vitro, CS-esposto contro-air esposto) alla 48 ore di post-esposizione sono stati confrontati con quelli di fumo sulla epiteliali bronchiali umane raccolte da broncoscopia e nasale epiteliale dal burshing turbinati inferiori (GSE16008 ) (in vivo, i fumatori vs fumatori) (B). Inserti, correlazione della espressione genica cambia tra la in vitro e in vivo dataset. Abbreviazioni: CS, fumo di sigaretta; NPA, perturbazione di ampiezza di rete.ove.com/files/ftp_upload/52325/52325fig5large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura.

Watch this Scientific Journal Video about Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke at JoVE.com

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Valutazione dell&#39;impatto di esposizione ripetuta di organotipica 3D bronchiali e nasali coltura tissutale modelli di Whole fumo di sigaretta

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

impact-assessment-repeated-exposure-organotypic-3d-bronchial-nasal

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Bioengineering

11.0K Views.  Philip Morris Products S.A.. The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Video: Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

Un protocollo per il rilevamento e scavenging in fase gassosa radicali liberi nel fumo di sigaretta Mainstream

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

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Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingegneria dei tessuti di un Umano 3D<em&gt; In vitro</em&gt; Sistema Tumore test

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Campione strategie di preparazione per la Spettrometria di Massa Imaging di 3D Models Cultura cellulare

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Multimateriali di cartilagine e pelle tessuto analoghi utilizzando un romanzo passivo unità tecnica di miscelazione per Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Un microcontrollore azionato il dispositivo per la generazione di estratti liquidi da Aerosol elettronico e fumo di sigaretta sigaretta convenzionale

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Valutazione elettrofisiologia cardiaca preclinica mediante mappatura ottica a doppia tensione e calcio delle fette cardiache di organitypica umana

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelli avanzati di fegato 3D per test di genotossicità in vitro a seguito dell'esposizione a lungo termine ai nanomateriali

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Una piattaforma tecnologica open source per la produzione di modelli di cultura 3D basati su idrogel in modo automatizzato e standardizzato

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

Isolamento e coltura di cellule epiteliali gengivali umane primarie utilizzando Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Generazione scalabile di sferoidi cellulari uniformi utilizzando un dispositivo di assemblaggio acustico 3D

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

Autore in primo piano: Sviluppo di un metodo di assemblaggio acustico senza scaffold per colture cellulari sferoidi 3D di alta qualità 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

Dispositivo di assemblaggio acustico tridimensionale per la produzione di massa di sferoidi cellulari

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...