Los autores desean agradecer a Maurice Smith y Marja Talikka para su revisión del manuscrito.

<ol>
	<li>Pezzulo, A. A., 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=The+air-liquid+interface+and+use+of+primary+cell+cultures+are+important+to+recapitulate+the+transcriptional+profile+of+in+vivo+airway+epithelia.">The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia.</a> American Journal of Physiology - Lung Cellular and Molecular Physiolog. 300, L25-L31 (2011).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nature reviews Molecular cell biolog. 8, 839-845 (2007).</li><li>Dvorak, A., Tilley, A. 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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Este estudio fue financiado por Philip Morris International.

Aquí, hemos demostrado la aplicabilidad de los modelos de tejidos nasales bronquial y organotípico humano para evaluar el impacto de la exposición repetida CS. Como alternativa a la experimentación con animales, se han desarrollado una serie de sistemas de exposición para las evaluaciones toxicológicas de la exposición al aerosol in vitro (por ejemplo., Vitrocell, Cultex, ALICE, etc.). Estos módulos de exposición también se pueden utilizar para una evaluación toxicológica de los contaminantes del aire, partículas en el aire, nanopartículas, etc. En este estudio, hemos utilizado el sistema Vitrocell que puede acomodar hasta 48 muestras diferentes de forma simultánea, lo que permite más grandes experimentos a escala y una menor variabilidad entre tratamientos. Para cada exposición al aerosol in vitro, el riesgo de contaminación de cultivo de tejidos sigue siendo un riesgo importante cuya mitigación exige un manejo cuidadoso de los cultivos de tejidos a lo largo de los experimentos. Medición de la deposición de partículas en tiempo real en la microbalanza durante el eexperimento Xposure permite monitorear que las dosis CS generados por el sistema de exposición son conformes a las expectativas. Para asegurar la exactitud de la deposición de partículas medido, creación de la medición en línea correctamente antes de la exposición es critial, por ejemplo, el establecimiento de la escala a 0. Además, debido a la diferencia entre las áreas de la microbalanza (2 cm) y la inserción de cultivo de tejido (0,33 cm 2), se ajustó el cálculo final de la zona de la inserción de cultivo: sólo el 33% de la deposición sobre la microbalanza refleja la deposición real en el inserto de cultivo. La medición de la TEER para determinar la funcionalidad de barrera apretado nudo y para evaluar la interrupción de la capa epitelial es un procedimiento relativamente fácil de implementar, que se informó aquí. Sin embargo, debido a que los modelos de tejidos bronquiales y nasales contienen células caliciformes intercalados productoras de moco, lavado apical debe realizarse antes de las meas TEERmedi-. El lavado apical es crítico debido a la presencia de la capa de moco y la variabilidad de su espesor puede sesgo de la medición de la TEER, interferring con el impacto de la exposición CS. Esta noción está de acuerdo con lo reportado por Hilgendorf y sus colegas, en el que la permeabilidad de las células Caco-2 se vio afectada por el co-cultivo con la línea de células caliciformes productoras de moco HT29 23. El moco necesita ser lavada antes de la medición TEER, porque el lavado apical justo antes de la exposición puede interferir con las respuestas de los tejidos a CS. Por lo tanto, la medición se lleva a cabo tres días antes de la exposición, y no justo antes de la exposición. Hemos demostrado que la actividad de CYP1A1 / CYP1B1 podría medirse a partir de los modelos de cultivos organotípicos tras la exposición CS aunque la actividad se incrementó sólo modestamente por CS. Esta señal débil puede ser amplificada por una incubación más largo del sustrato CYP1A1 / 1B1 (es decir., Luciferina-CEE), por ejemplo para24 h (datos no mostrados). Una de las limitaciones de la medición de la actividad de CYP en el presente trabajo es la ausencia de normalización a nivel de la proteína CYP o para el recuento de células, lo que podría ser tomada en consideración para futuros estudios para garantizar que la alteración en la actividad de la enzima no se ve influenciada por la alteración de ya sea el nivel de proteína o el recuento de células. Nos informó que la exposición CS inhibe la ciliar superando tanto en los modelos de tejido bronquial y nasal. Observaciones similares se realizaron en diversos modelos de mamíferos y no mamíferos 12. Para ciliar batiendo medición, asegurando que los tejidos son manipulados y tratados de manera similar es crítica, por ejemplo, si se implementa el cambio medio, debe ser aplicado en todas las muestras. Sutto y sus colegas informaron que el pH afecta la ciliar mamíferos latiendo frecuencia 24. Por lo tanto, al comparar superando frecuencias entre las células tratadas con diferentes COMPUESTOS / mezclas, pHajuste debe ser considerado para minimizar la variabilidad de la medición latido ciliar. Además, la temperatura a la que se lleva a cabo la medición es también crítico como la frecuencia de golpeo ciliar disminuye con la disminución de la temperatura. Para minimizar la variabilidad debida a estos cambios, una incubadora de etapa superior, equipadas con la temperatura, CO2 y de la humedad se utilizó en este estudio. A pesar de ello, se observó que el ciliar batiendo frecuencias en los tejidos bronquiales después de la exposición CS eran muy variables (es decir., Aumentar en un inserto y disminuir en el otro inserto), lo que sugiere que el movimiento ciliar es correcto altamente perturbado después de la exposición. En contraste, se observó la ausencia de frecuencias de batido ciliar medibles en el tejido nasal después de la exposición CS, lo que sugiere que la respuesta del tejido nasal es más sensible y consistente. Esto está de acuerdo con una publicación anterior que demuestra que el tejido de la nariz tiene una menor capacidad para desintoxicar comoen comparación con el bronquio 25. Por último, hemos demostrado que la expresión de genes de perfiles del bronquial organotípico y modelos de tejidos nasales expuestos a CS podría demostrar un impacto de CS en el metabolismo de xenobióticos. Curiosamente, la alteración observada en el metabolismo de xenobióticos en el organotípicos bronquial y nasal en modelos in vitro expuestos a CS asemejan a la de la situación in vivo en los fumadores como se discute en mayor detalle en una publicación anterior 15. Para los análisis de la expresión génica, usando el instrumento robótico hace un análisis de alto rendimiento posible. Por otra parte, el manejo robótico automático aumenta aún más la consistencia y exactitud de los resultados de la expresión génica. Sin embargo, la recogida rápida de las muestras de tejido era crítico para evitar la degradación del ARN durante la extracción de RNA. El procesamiento del ARN y los enfoques transcriptomic descritos aquí también se pueden aplicar a in vivo muestras de tejido.

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.

En los resultados se describen a continuación, los tejidos organotípicos fueron células epiteliales humanas primarias aisladas de saludable, no fumar, donantes de raza blanca y se reconstituyeron utilizando fibroblastos 15. Modelos de tejidos nasales bronquial 3D y En bronquial vitro y modelos organotípicos nasales parecerse a nivel celular del epitelio del tracto respiratorio humano (Figura 2). Exposición CS Modelos de tejidos nasales bronquial organotípicos y pueden estar expuestos repetidamente a la corriente principal de CS en la interfase aire-líquido. El procedimiento experimental utilizado para la exposición al humo de los resultados ilustrados aquí se representa en la figura 1. Grabación de movimiento ciliar Los cilios golpiza fue grabado en los tejidos expuestos al aire, como se ilustra en los videos. La exposición CS se asoció con una reducción de la frecuencia de latido ciliar: el pico más fuerte observado alrededor4 Hz en el simulacro expuestas y antes de la exposición no se observa por más tiempo después de la exposición CS (Figura 3). Esta frecuencia reducida haría los cilios latir menos eficiente para eliminar el moco y agentes infecciosos 22. La medición de la TEER y CYP1A1 / actividad 1B1 TEER se midió 48 horas después de la última exposición a garantizar el tejido sigue siendo funcional. CYP1A1 / 1B1 actividad también se midió 48 h después del final de la exposición como se ilustra en la Figura 1. Los valores de TEER en los tejidos expuestos-CS no fueron significativamente diferentes en comparación con los tejidos expuestos al aire, confirmando que no hay ningún efecto citotóxico principal de humo a esta concentración. En la 48 h después de la exposición, CYP1A1 / 1B1 actividad de los tejidos se incrementó ligeramente, lo que indica un aumento modesto del mecanismo de defensa del tejido después de la exposición (Figura 4). Los perfiles de expresión génica y sistemas basados ​​en la red bilogía enfoque para estudiar el impacto de la exposición CS en la alteración del metabolismo de xenobióticos Los tejidos se recogieron a las 48 h puntos de tiempo después de la exposición. Posteriormente, se llevó a cabo el análisis de microarrays para generar perfiles de expresión génica de la CS- y tejidos expuestos al aire. Impacto de la exposición CS sobre el metabolismo de xenobióticos se investigó mediante un enfoque de la biología de sistemas basados ​​en la red que pueda traspasarse de los perfiles de expresión génica y como se informó anteriormente 15. Utilizando un enfoque de biología de sistemas basados ​​en la red muestra que los impactos de exposición CS en el nivel de los nodos de red troncal en el modelo de red de metabolismo de xenobióticos estaban bien correlacionados entre la in vitro y en conjuntos de datos in vivo (Figura 5). En contraste, en el nivel de cada individuo cambios de expresión génica, se observaron correlaciones entre el pobres in vitro (CS expuestos al aire vs. expuesta) e in vivo (fumadores vs. no fumadores). Figura 1. Esquema de procedimiento de la exposición repetida de CS a los modelos de tejidos bronquiales organotypic Abreviaturas:. CYP, citocromo P450; CS, el humo del cigarrillo; TEER, resistencia eléctrica transepitelial. <img alt="Figura 2" src="/files/ftp_upload/52325/52325fig2.jpg" /> Figura 2. organotípicos bronquial y modelos de cultivo de tejidos epiteliales nasales. Historieta que ilustra la bronquial (A) y el inserto de cultivo nasal (B) tejido en la interfase aire-líquido. Los modelos in vitro contenían células ciliadas que se muestran en la capa apical de las células teñidas con hematoxilina y eosina (C durante bronquial, nasal para D) como se informó anteriormente 15. Los modelos fueron co-cultivadas con fibroblastos que son importantes<html> para el crecimiento y diferenciación de células epiteliales (indicado por las flechas). La tinción de los marcadores de linaje de las vías respiratorias: p63 (E para bronquial, F para nasal) y Muc5AC (G para bronquial, H para nasal) se muestran como se informó anteriormente 15. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52325/52325fig3.jpg" /> Figura 3. Los cilios Beating frecuencia. Cilios frecuencia de latidos (CBF) se midió en aire expuesto y en el cultivo antes y después de la exposición. La disminución de la CBF fue visto después de la exposición CS (resultados representativos de dos inserto se muestran como réplica 1 (R1) y se replican 2 (R2)). ad / 52325 / 52325fig4.jpg &quot;/&gt; Figura 4. Medición de CYP1A1 / actividad CYP1B1 y TEER paneles de izquierda. Indican la actividad CYP1A1 / CYP1B1 en el bronquial (A) y nasal (b) modelos de tejido. Paneles de la derecha indican la medición TEER en el bronquial (C) y nasal (D) los modelos de tejido. Se muestran las medias ± SD. Abreviaturas: CS, el humo del cigarrillo; CYP, el citocromo P450; TEER, resistencia eléctrica transepitelial; RLU, unidad de luminiscencia relativa. <img alt="Figura 5" src="/files/ftp_upload/52325/52325fig5.jpg" /> Figura 5. Los sistemas basados ​​en la red enfoque de la biología de la evaluación de impacto de la exposición al CS en el contexto del metabolismo de xenobióticos. Los cambios de los niveles de expresión de los genes se utilizaron para calcular la activación del nodo columna vertebral del modelo de red xenobióticos Metabolismo (A) </<html> strong&gt; como se informó en una publicación previa 15. La figura de la derecha ilustra el concepto de la cuantificación de los nodos troncales, también conocidos como &quot;valor de la red troncal diferencial&quot;, utilizando el enfoque de NPA. Los óvalos azules representan la actividad de los nodos de la red principal (es decir., La capa funcional) y las bolas verdes representan la expresión de genes (es decir., La capa de la transcripción). Impactos de la exposición CS en los tejidos bronquiales organotypic (in vitro, CS-expuestos vs aire expuesto) a las 48 h de post-exposición se compararon con los de fumar en epitelio bronquial humano recogido por broncoscopia y nasal epitelial por burshing cornete inferior (GSE16008 ) (in vivo, los fumadores frente a los no fumadores) (B). Inserciones, correlación de la expresión génica cambios entre la in vitro e in vivo de datos. Abreviaturas: CS, el humo del cigarrillo; NPA, red amplitud perturbación.ove.com/files/ftp_upload/52325/52325fig5large.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta 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.

Evaluación del Impacto de la exposición repetida de Organotípicos 3D bronquiales y nasales Modelos Tissue Culture Whole humo del cigarrillo

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

Research

JoVE Journal

Bioengineering

11.0K vistas.  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 protocolo para la detección y borrado en fase gaseosa radicales libres en el humo del cigarrillo 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.  ...

Investigación

Bioingeniería

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.

Ingeniería de tejidos de un humano 3D<em&gt; In vitro</em&gt; Sistema de Prueba de Tumores

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.

Preparación de la muestra Estrategias para Espectrometría de Masas de imágenes de modelos de cultivo celular en 3D

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.

Bioprinting de cartílago y piel análogos de tejido utilizando un pasivo novela mezcla de unidad técnica de 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 microcontrolador funciona dispositivo para la generación de extractos líquidos de Aerosol de humo y electrónicos cigarrillo cigarrillo convencional

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

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.

Modelos avanzados de hígado 3D para pruebas de genotoxicidad in vitro tras la exposición a nanomateriales a largo plazo

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 plataforma tecnológica de código abierto para fabricar modelos de cultivo 3D basados en hidrogel de manera automatizada y estandarizada

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.

Aislamiento y cultivo de células epiteliales gingivales humanas primarias utilizando 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 ...

Generación escalable de esferoides de célula uniforme mediante un dispositivo de ensamblaje acústico 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.

Autor destacado: Desarrollo de un método de ensamblaje acústico sin andamios para el cultivo de esferoides celulares 3D de alta calidad 

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 de montaje acústico tridimensional para la fabricación en masa de esferoides celulares

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