Os autores gostariam de agradecer a Maurice Smith e Marja Talikka para a sua revisão do manuscrito.

<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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Este estudo foi financiado pela Philip Morris International.

Aqui, nós têm demonstrado a aplicabilidade de modelos de tecido nasal e bronquial organotípicas humanas para avaliar o impacto da exposição repetida CS. Como uma alternativa para testes em animais, foram desenvolvidas uma série de sistemas de exposição para as avaliações toxicológicas de exposição ao aerossol in vitro (eg., Vitrocell, Cultex, ALICE, etc.). Estes módulos de exposição também pode ser utilizado para uma avaliação toxicológica dos poluentes transportados pelo ar, partículas em suspensão, nanopartículas, etc. Neste estudo, foi utilizado o sistema Vitrocell que pode acomodar até 48 amostras diferentes simultaneamente, permitindo experiências de maior escala e menor variabilidade entre tratamentos. Para cada exposição ao aerossol in vitro, o risco de contaminação da cultura de tecido continua a ser um grande risco cuja atenuação exige uma manipulação cuidadosa das culturas de tecido durante todo o experimento. Medindo a deposição de partículas em tempo real sobre o microbalança de e durante oXposure experimento permite monitorar se as doses CS geradas a partir do sistema de exposição estão em conformidade com as expectativas. Para garantir a precisão da deposição de partículas medida, estabelecendo-se a medição on-line corretamente antes da exposição é critial, por exemplo, a criação de escala a 0. Além disso, por causa da diferença entre as áreas da microbalança (cm 2) e o inserção de cultura de tecido (0,33 cm 2), foi ajustado o cálculo final para a área da inserção de cultura: apenas 33% da deposição na microbalança reflectir a deposição efectiva na lâmina de cultura. Medição da TEER verificar funcionalidade barreira tight-junção e avaliar rompimento da camada epitelial é um procedimento relativamente fácil de implementar, o que nós informamos aqui. No entanto, porque os modelos de tecido brônquico e nasal contém células caliciformes intercaladas produtoras de muco, lavagem apical precisa ser realizada antes dos meas TEERmedi-. A lavagem apical é crítica, pois a presença da camada de muco e a variabilidade da sua espessura pode enviesar a medição de TEER, ou seja, interferindo com o impacto da exposição CS. Esta noção está de acordo com o que foi relatado por Hilgendorf e colegas, em que a permeabilidade de células Caco-2 foi afectada pela co-cultura com a linha de células caliciformes produtoras de muco HT29 23. O muco precisa ser lavado antes da medição TEER, porque lavagem apical à direita antes da exposição pode interferir com as respostas teciduais para CS. Por conseguinte, a medição é realizada três dias antes da exposição, e não antes da exposição à direita. Nós mostramos que a atividade CYP1A1 / CYP1B1 pode ser medida a partir dos modelos de cultura organotypic seguintes exposição CS embora a atividade foi apenas modestamente aumentou CS. Este sinal fraco pode ser amplificado por um mais longo de incubação do substrato de CYP1A1 / 1B1 (ie., Luciferina-CEE), por exemplo, para24 horas (dados não mostrados). Uma das limitações da medição da actividade CYP no presente trabalho é a ausência de normalização ao nível da proteína CYP ou para a contagem de células, o que pode ser tomado em conta para futuros estudos para assegurar que a alteração na actividade da enzima não é influenciada pela alteração do nível ou da proteína ou a contagem de células. Nós relataram que a exposição CS inibiu o batimento ciliar, tanto na modelos de tecido dos brônquios e nasal. Observações semelhantes foram feitas em diversos mamíferos e não mamíferos modelos 12. Para ciliar batendo medição, assegurando que os tecidos são tratados e tratados de um modo semelhante é crítico, por exemplo, se a mudança meio é realizada, deverá ser aplicado para todas as amostras. Sutto e colegas relataram que afetou o pH ciliar mamíferos batendo frequência 24. Assim, quando se comparam batendo frequências entre as células tratadas com diferentes misturas / compunds, pHDeve ser considerado ajuste para minimizar a variabilidade da medição da batimentos ciliares. Além disso, a temperatura na qual a medição é conduzida também é crítico que a frequência de batimento ciliar gotas com a diminuição da temperatura. Para minimizar a variabilidade devido a essas mudanças, uma incubadora fase de topo, equipados com a temperatura, CO2, e para controlo de humidade foi usada neste estudo. Apesar disso, observou-se que o batimento ciliar freqüências nos tecidos bronquiais após a exposição CS foram altamente variável (ie., Aumentar em um insert e diminuir na outra inserção), sugerindo que o batimento ciliar é certo altamente perturbado após a exposição. Em contraste, observou-se a ausência de frequências de batimento ciliar mensuráveis ​​no tecido nasal após exposição CS, sugerindo que a resposta do tecido nasal é mais sensível e mais consistente. Isto está de acordo com uma publicação anterior mostrando que o tecido nasal tem uma menor capacidade de desintoxicar comoem comparação com o brônquio 25. Finalmente, mostrou-se que a expressão do gene a partir do perfil de modelos de tecido nasal e bronquial organotípicas expostos a CS poderiam demonstrar um impacto de CS no metabolismo xenobiótico. Curiosamente, a alteração observada no metabolismo xenobiótico no brônquica organotípicas e nasal em modelos in vitro expostos a CS assemelhar-se à situação in vivo em fumadores como discutido em maior detalhe numa publicação anterior 15. Para análises de expressão de genes, utilizando o instrumento robotizado faz uma análise de alto rendimento possível. Além disso, a manipulação robótica automática aumenta ainda mais a consistência ea precisão dos resultados de expressão gênica. No entanto, a recolha rápida das amostras de tecido foi fundamental para evitar a degradação de RNA durante a extracção de ARN. O processamento de RNA e abordagens transcriptómico descritos aqui também podem ser aplicados a amostras de tecido 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.

Em resultados descritos a seguir, os tecidos eram organotypic células epiteliais humanas primárias isoladas de saudável, não-fumadores, doadores caucasianos e foram reconstituídos usando fibroblastos 15. Os modelos de tecido nasal e bronquial 3D Em brônquica vitro e modelos organotípicas nasais assemelham ao nível celular do epitélio do tracto respiratório humano (Figura 2). Exposição CS Os modelos de tecido nasal e bronquial organotípicas pode ser repetidamente exposta a corrente principal CS na interface ar-líquido. O procedimento experimental exposição ao fumo utilizados para os resultados ilustrados aqui está representado na Figura 1. Gravação do batimento ciliar O batimento ciliar foi gravado nos tecidos expostos ao ar, como ilustrado nos vídeos. Exposição CS foi associado a uma redução da frequência de batimento ciliar: o pico mais forte observada em torno4 Hz na farsa exposto antes da exposição e não é observado por mais tempo após a exposição ao CS (Figura 3). Esta frequência reduzida faria os cílios batendo menos eficiente para remover o muco e agentes infecciosos 22. Medição da TEER e CYP1A1 atividade / 1B1 TEER foi medida 48 horas após a última exposição a assegurar o tecido ainda está funcional. CYP1A1 / 1B1 actividade também foi medida 48 h após terminada a exposição, como se ilustra na Figura 1. Os valores TEER nos tecidos CS-expostos não foram significativamente diferentes quando comparados com os tecidos expostos ao ar confirmando que não há efeito citotóxico principal de fumo a esta concentração. No 48 h pós-exposição, CYP1A1 / 1B1 actividade dos tecidos foi ligeiramente aumentada, indicando um aumento modesto do mecanismo de defesa do tecido após a exposição (Figura 4). Gene perfis de expressão e sistemas baseados em rede biologia abordagem para estudar o impacto da exposição CS sobre a alteração do metabolismo de xenobióticos Os tecidos foram coletados 48 horas após a exposição pontos de tempo. Posteriormente, a análise de microarray foi conduzido para gerar perfis de expressão gênica do CS- e tecidos expostos ao ar. Impacto da exposição CS sobre o metabolismo de xenobióticos foi investigada através de uma abordagem de biologia de sistemas baseados em rede alavancada a partir dos perfis de expressão genética e como foi dito anteriormente 15. Usando uma abordagem de biologia de sistemas à base de rede mostra que os impactos da exposição CS ao nível da espinha dorsal nós no modelo de rede metabolismo de xenobióticos foram bem correlacionados entre o in vitro e in vivo em conjuntos de dados (Figura 5). Em contraste, a nível de cada indivíduo mudanças de expressão genética, foram observadas correlações pobres entre a in vitro (CS-expostos vs. exposto ao ar) e in vivo (fumantes vs. não-fumantes). Figura 1. procedimento esquemático da exposição repetida de CS para os modelos organotypic tecidos bronquiais Abreviaturas:. CYP, citocromo P450; CS, fumaça de cigarro; TEER, resistência elétrica transepitelial. <img alt="Figura 2" src="/files/ftp_upload/52325/52325fig2.jpg" /> Figura 2. organotípicas brônquica e modelos de cultura de tecidos epiteliais nasais. Dos desenhos ilustra o brônquica (A) e nasal (B) de cultura de tecidos de inserção na interface ar-líquido. Os modelos in vitro continha células ciliadas mostrados na camada apical das células coradas hematoxilina e eosina (C para brônquico, nasal para D), tal como relatado anteriormente 15. Os modelos foram co-cultivadas com fibroblastos que são importantes<html> para o crescimento e diferenciação de células epiteliais (indicado pelas setas). Coloração de marcadores de linhagem das vias aéreas: p63 (E para brônquica, F para nasal) e MUC5AC (G para brônquica, H para nasal) são mostrados como relatado anteriormente 15. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52325/52325fig3.jpg" /> Figura 3. O batimento ciliar Frequency. Cilia freqüência de batimento (CBF) foi medido em expostos ao ar e na cultura antes e após a exposição. Diminuição da CBF, após exposição CS (resultados representativos de dois insert são mostrados como replicar 1 (R1) e replicar 2 (R2)). ad / 52325 / 52325fig4.jpg &quot;/&gt; Figura 4. Medição da CYP1A1 atividade e TEER / CYP1B1. Pannels esquerda indicam a atividade de CYP1A1 / CYP1B1 no nasal (B) Os modelos de tecido dos brônquios (A) e. Painéis da direita indicam a medição TEER no brônquica (C) e nasal (D) Os modelos de tecido. Meios ± SD são mostrados. Abreviaturas: CS, fumaça de cigarro; CYP, do citocromo P450; TEER, resistência elétrica transepitelial; RLU, unidade luminescense relativa. <img alt="Figura 5" src="/files/ftp_upload/52325/52325fig5.jpg" /> Figura 5. Foram utilizados sistemas baseados em rede biologia abordagem para a avaliação do impacto da exposição CS no contexto de metabolismo xenobiótico. As alterações dos níveis de expressão dos genes para calcular a activação de nó do eixo principal do modelo de rede do metabolismo xenobiótico (A) </<html> strong&gt; como relatado na publicação anterior 15. A figura à direita ilustra o conceito de quantificação dos nós de backbone, também conhecido como &quot;valor backbone da rede diferencial&quot;, usando a abordagem NPA. As ovais azuis representam a actividade dos nós de esqueleto (isto é., A camada funcional) e as esferas verdes representam a expressão de genes (isto é., A camada de transcrição). Impactos da exposição CS nos tecidos bronquiais organotypic (in vitro, CS-expostos vs. exposto ao ar) foram comparados ao 48 h de pós-exposição aos do tabagismo sobre epitelial brônquica humano coletado por broncoscopia e nasal epitelial por burshing corneto inferior (GSE16008 ) (in vivo, os fumantes contra os não-fumantes) (B). As inserções, a correlação da expressão do gene muda entre o in vitro e em conjuntos de dados in vivo. Abreviaturas: CS, fumaça de cigarro; NPA, perturbação de amplitude rede.&quot;target =&quot; _ ove.com/files/ftp_upload/52325/52325fig5large.jpg blank &quot;&gt; Clique aqui para ver uma versão maior desta 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.

Avaliação do Impacto da exposição repetida de Organotípicas 3D brônquica e cultura nasal Tissue Models para Whole fumo do cigarro

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 visualizações.  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.

Vídeo: 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.

Um Protocolo para Detecção e eliminação em fase gasosa radicais livres do fumo do cigarro 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.  ...

Bioengenharia

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.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

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.

Amostra Estratégias Preparação para Mass Spectrometry imagem da cultura celular 3D Models

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 da cartilagem e Analogs de tecido de pele utilizando um romance passiva unidade técnica de mistura para 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.

Um microcontrolador operado dispositivo para a geração de extratos líquidos de aerossol de cigarro eletrônico e fumo cigarro convencional

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.

Avaliação de Eletrofisiologia Cardíaca Pré-Plínica por Mapeamento Óptico de Dupla Tensão e Cálcio de Fatias Cardíacas Organotípicas Humanas

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.

Modelos avançados de fígado 3D para testes de genotoxicidade in vitro após exposição nanomaterial de longo prazo

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.

Uma plataforma de tecnologia de código aberto para fabricar modelos de cultura 3D à base de hidrogel de forma automatizada e padronizada

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 Cultura das Células Epiteliais Gengivais Humanas Primárias usando 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 ...

Geração escalável de esferoides de células uniformes usando um dispositivo de montagem acústica 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 em destaque: Desenvolvimento de um método de montagem acústica sem andaimes para cultura de esferóides de células 3D de alta qualidade 

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 Montagem Acústica Tridimensional para Fabricação em Massa de Células Esferoides

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