Les auteurs tiennent à remercier Maurice Smith et Marja Talikka pour la révision du manuscrit.

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Cette étude a été financée par Philip Morris International.

Ici, nous avons démontré l&#39;applicabilité de bronchique organotypique humaine et modèles de tissus nasaux pour évaluer l&#39;impact d&#39;une exposition répétée CS. Comme une alternative à l&#39;expérimentation animale, un certain nombre de systèmes d&#39;exposition ont été élaborées pour les évaluations toxicologiques de l&#39;exposition d&#39;aérosol in vitro (par exemple., Vitrocell, Cultex, ALICE, etc.). Ces modules d&#39;exposition peuvent également être utilisés pour une évaluation toxicologique des polluants atmosphériques, les particules en suspension, les nanoparticules, etc. Dans cette étude, nous avons utilisé le système Vitrocell qui peut accueillir jusqu&#39;à 48 échantillons différents simultanément, ce qui permet pour les grandes expériences à grande échelle et une plus faible variabilité entre les traitements. Pour chaque exposition aérosol in vitro, le risque de contamination de la culture de tissu demeure un risque majeur dont l&#39;atténuation exige une manipulation soignée des cultures de tissus tout au long des expériences. Mesure de dépôt de particules en temps réel sur le cours de la microbalance électroniquexposition expérience permet de surveiller que les doses CS générés par le système d&#39;exposition sont conformes aux attentes. Pour assurer la précision du dépôt de particule mesurée, mise en place correctement la mesure en ligne avant l&#39;exposition est critial, par exemple, la mise en place de l&#39;échelle à 0. En outre, en raison de la différence entre les zones de la microbalance (cm 2) et le insert de culture de tissu (0,33 cm 2), on a ajusté le calcul final de la zone de l&#39;insert de culture: seulement 33% du dépôt sur ​​la microbalance reflète le dépôt effectif dans l&#39;insert de culture. Mesure de TEER de vérifier la fonctionnalité de barrière étanche jonction et d&#39;évaluer la perturbation de la couche épithéliale est une procédure relativement facile à mettre en œuvre, que nous avons rapporté ici. Cependant, parce que les modèles de tissus bronchiques et nasales les contiennent des cellules caliciformes intercalées mucus production, lave-apicale doit être effectuée avant que les AME TEERme-. Le lavage apicale est essentiel parce que la présence de la couche de mucus et la variabilité de son épaisseur peut biaiser la mesure de TEER, interférer avec l&#39;impact de l&#39;exposition CS. Cette notion est en accord avec ce qui a été rapporté par Hilgendorf et ses collègues, dans lequel la perméabilité des cellules Caco-2 a été affectée par la co-culture avec la lignée cellulaire gobelet mucus produisant HT29 23. Le mucus doit être lavé avant la mesure TEER, parce laver apicale juste avant l&#39;exposition peut interférer avec les réponses tissulaires à CS. Par conséquent, la mesure est effectuée trois jours avant l&#39;exposition, et non juste avant l&#39;exposition. Nous avons montré que l&#39;activité CYP1A1 / CYP1B1 pourrait être mesurée à partir des modèles de culture organotypiques après exposition CS bien que l&#39;activité a été que modestement augmenté de CS. Ce signal faible peut être amplifié par une incubation plus longue du substrat CYP1A1 / de 1B1 (ie., La luciférine-FEC), par exemple pour24 heures (données non présentées). Une des limites de la mesure de l&#39;activité de CYP dans le présent travail est l&#39;absence de normalisation au niveau de la protéine CYP ou de la numération cellulaire, qui pourraient être prises en considération pour de futures études pour se assurer que la modification de l&#39;activité enzymatique ne est pas influencée par la modification de l&#39;une ou l&#39;autre niveau de la protéine ou du nombre de cellules. Nous avons signalé que l&#39;exposition CS a inhibé la ciliaire battre à la fois dans l&#39;nasale et modèles de tissus bronchiques. Des observations similaires ont été faites dans des modèles différents mammifères et non mammifères 12. Pour battement ciliaire mesure, en veillant à ce que les tissus sont manipulés et traités d&#39;une manière similaire est critique, par exemple si le changement moyen est mis en oeuvre, il doit être appliqué pour tous les échantillons. Sutto et ses collègues ont rapporté que le pH affecté le ciliaire mammifère battant fréquence 24. Ainsi, lorsque l&#39;on compare battant fréquences entre les cellules traitées avec différentes compunds / mélanges, pHajustement doit être considérée pour réduire au minimum la variabilité de la mesure de battements ciliaires. En outre, la température à laquelle la mesure est effectuée est également essentiel que la fréquence de battement ciliaire diminue lorsque la température diminue. Pour minimiser la variabilité due à ces changements, un incubateur stade supérieur, équipées avec la température, de CO 2, et contrôle de l&#39;humidité a été utilisé dans cette étude. Malgré cela, nous avons observé que le battement ciliaire fréquences dans les tissus bronchiques après l&#39;exposition CS était très variable (ie., Augmenter en un insert et diminuer dans l&#39;autre insert), ce qui suggère que le battement ciliaire est juste très perturbé après l&#39;exposition. En revanche, nous avons observé l&#39;absence d&#39;mesurables fréquences de battement ciliaire dans le tissu nasal après exposition CS, ce qui suggère que la réponse du tissu nasal est plus sensible et cohérente. Ce est en accord avec une précédente publication montrant que le tissu nasal a une capacité inférieure à détoxifier quepar rapport à 25 la bronche. Enfin, nous avons montré que l&#39;expression du gène de profilage de la bronchique organotypique et modèles de tissus nasaux exposés à CS pourrait démontrer un impact de CS sur le métabolisme des xénobiotiques. Fait intéressant, la modification observée dans le métabolisme des xénobiotiques dans le bronchique et nasale organotypique des modèles in vitro exposé à CS qui ressemblent à de la situation in vivo chez les fumeurs comme on le verra plus en détail dans une publication précédente 15. Pour les analyses de l&#39;expression génique, en utilisant l&#39;instrument robotisé fait une analyse à haut débit possible. De plus, la manipulation robotisée automatique augmente encore la cohérence et la précision des résultats d&#39;expression de gène. Néanmoins, la collecte rapide des échantillons de tissu est critique pour éviter la dégradation de l&#39;ARN au cours de l&#39;extraction de l&#39;ARN. Le traitement de l&#39;ARN et des approches transcriptomiques décrits ici peuvent également être appliqués à des échantillons de tissus 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.

Dans les résultats décrits ci-dessous, les tissus étaient organotypiques cellules épithéliales humaines primaires isolées de santé, non-fumeurs, des donateurs et du Caucase ont été reconstitués en utilisant des fibroblastes 15. Bronchique 3D et modèles de tissus nasaux Dans bronchique vitro et modèles organotypiques nasales ressemblent au niveau cellulaire de l&#39;épithélium des voies respiratoires humaines (Figure 2). L&#39;exposition CS Bronchique organotypique et modèles de tissus nasaux peuvent être exposés de façon répétée à intégrer CS à l&#39;interface air-liquide. La procédure expérimentale de l&#39;exposition à la fumée utilisée pour les résultats illustrés ici est représenté sur la figure 1. Enregistrement du battement ciliaire Cilia battement a été enregistrée dans les tissus exposés à l&#39;air, comme illustré dans les vidéos. CS exposition a été associée à une réduction de la fréquence de battement ciliaire: le plus fort pic observé autour4 Hz dans le simulacre exposé et avant l&#39;exposition ne est pas observée plus longtemps après l&#39;exposition CS (Figure 3). Cette fréquence réduite rendrait les cils battant moins efficace pour éliminer le mucus et les agents infectieux 22. Mesure de TEER et CYP1A1 activité / 1B1 TEER a été mesurée 48 h après la dernière exposition pour assurer le tissu est encore fonctionnel. CYP1A1 / 1B1 activité a également été mesurée 48 h après la fin de l&#39;exposition comme illustré sur la Figure 1. Les valeurs TEER dans les tissus CS-exposées étaient pas significativement différentes par rapport aux tissus de l&#39;air exposé confirmant qu&#39;il n&#39;y a pas d&#39;effet cytotoxique important de fumée à cette concentration. Au post-exposition 48 h, CYP1A1 / 1B1 activité des tissus a été légèrement augmenté, indiquant une modeste augmentation de mécanisme de défense de tissu après l&#39;exposition (Figure 4). Gene profils d&#39;expression et des systèmes basés sur le réseau biologie approche pour étudier l&#39;impact de l&#39;exposition CS sur l&#39;altération du métabolisme des xénobiotiques Les tissus ont été prélevés à des moments post-exposition 48 h. Par la suite, l&#39;analyse de puces à ADN a été réalisée pour générer des profils d&#39;expression génique de la CS et des tissus exposés à l&#39;air. Impact de l&#39;exposition sur le métabolisme des xénobiotiques CS a été étudiée en utilisant une approche basée sur le réseau biologie des systèmes à effet de levier à partir des profils d&#39;expression génique et comme rapporté plus tôt 15. En utilisant une approche biologie des systèmes en réseau montre que les effets de l&#39;exposition CS au niveau des nœuds de réseau fédérateur dans le modèle de réseau de métabolisme des xénobiotiques étaient bien corrélées entre le in vitro et in vivo des ensembles de données (figure 5). En revanche, au niveau de chacun des changements d&#39;expression génique, le manque de corrélation n&#39;a été observée entre le in vitro (CS-exposé vs. air-exposée) et in vivo contre les non-fumeurs (fumeurs). Figure 1. Représentation schématique de la procédure exposition répétée de CS pour les modèles de tissus bronchiques organotypiques Abréviations:. CYP, cytochrome P450; CS, la fumée de cigarette; TEER, résistance électrique transépithéliale. <img alt="Figure 2" src="/files/ftp_upload/52325/52325fig2.jpg" /> Figure 2. organotypiques bronchique et les modèles de culture de tissus épithéliales nasales. Dessinée illustrant le bronchique (A) et nasal (B) la culture tissulaire insert à l&#39;interface air-liquide. Les modèles in vitro contiennent des cellules ciliées indiquées dans la couche apicale des cellules colorées hématoxyline &amp; éosine (C pendant bronchique, D) pour l&#39;administration nasale 15 comme indiqué précédemment. Les modèles ont été co-cultivées avec des fibroblastes qui sont importantes<html> pour la croissance et la différenciation des cellules épithéliales (indiqué par des flèches). Coloration des marqueurs de la lignée des voies respiratoires: p63 (E pour bronchique, F pour nasale) et MUC5AC (G pour bronchique, H pour nasale) sont présentés comme indiqué précédemment 15. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">Se il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 3" src="/files/ftp_upload/52325/52325fig3.jpg" /> Figure 3. Fréquence Battre Cilia. Cilia fréquence de battement (CBF) a été mesuré dans l&#39;air et exposées dans la culture avant et après l&#39;exposition. Une diminution de la CBF a été observée après l&#39;exposition CS (résultats représentatifs de deux insert sont présentés comme une réplique (R1) et répliquent 2 (R2)). ad / 52325 / 52325fig4.jpg &quot;/&gt; Figure 4. Mesure de l&#39;activité CYP1A1 / CYP1B1 et TEER. Panneaux gauche indiquent l&#39;activité CYP1A1 / bronchique dans la CYP1B1 (A) et nasal (B) modèles de tissus. Panneaux de droite indiquent la mesure de la TEER bronchique (C) et nasal (D) modèles de tissus. Moyens ± SD sont présentés. Abréviations: CS, la fumée de cigarette; CYP, cytochrome P450; TEER, résistance électrique transépithélial; AVC, unité de luminescense relative. <img alt="Figure 5" src="/files/ftp_upload/52325/52325fig5.jpg" /> Figure 5. Les systèmes basés sur le réseau approche de biologie pour l&#39;évaluation de l&#39;exposition CS dans le cadre du métabolisme des xénobiotiques de l&#39;impact. Les changements de niveaux d&#39;expression des gènes ont été utilisés pour calculer l&#39;activation du noeud épine dorsale du modèle de réseau Xénobiotique métabolisme (A) </<html> strong&gt; tel que rapporté dans une publication précédente 15. La figure de droite illustre le concept de la quantification des noeuds du squelette, aussi connu comme &quot;valeur de réseau fédérateur différentiel», en utilisant l&#39;approche NPA. Les ovales bleues représentent l&#39;activité des noeuds de réseau fédérateur (par exemple., La couche fonctionnelle) et les billes vertes représentent l&#39;expression des gènes (ce est à dire., La couche de transcription). Impacts d&#39;une exposition dans les tissus bronchiques CS organotypiques (in vitro, CS-exposés vs exposées à l&#39;air) au 48 h de post-exposition ont été comparés à ceux du tabagisme sur l&#39;épithélium bronchique humain prélevé par bronchoscopie et nasale épithéliale par burshing cornet inférieur (GSE16008 ) (in vivo, les fumeurs vs les non-fumeurs) (B). Encarts, la corrélation de l&#39;expression du gène change entre le in vitro et in vivo des ensembles de données. Abréviations: CS, la fumée de cigarette; NPA, l&#39;amplitude de perturbation du réseau.ove.com/files/ftp_upload/52325/52325fig5large.jpg &quot;target =&quot; _ blank &quot;&gt; Se il vous plaît cliquer ici pour voir une version plus grande de cette figure.

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.

Évaluation de l&#39;impact de l&#39;exposition répétée de organotypiques 3D bronchiques et nasales Tissue Culture modèles Whole fumée de cigarette

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 vues.  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.

Vidéo: 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 protocole pour la détection et de nettoyage en phase gazeuse des radicaux libres dans la fumée de cigarette Mainstream

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

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Bio-ingénierie

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.

génie tissulaire d&#39;un être humain 3D<em&gt; In vitro</em&gt; Système de tumeur de test

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

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

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

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

Exemple Stratégies Préparation de Spectrométrie de Masse Imagerie de culture cellulaire 3D Modèles

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 du Cartilage et peau tissus analogues utilisant un roman passif unité Technique de mélange pour 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 microcontrôleur exploité le dispositif pour la génération d’extraits liquides d’aérosol de Cigarette fumée et électronique de Cigarette classique

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.

Évaluation de l’électrophysiologie cardiaque préclinique par la double tension et la cartographie optique du calcium des tranches cardiaques organotypic humaines

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.

Modèles hépatiques 3D avancés pour les tests de génotoxicité in vitro après une exposition à long terme aux nanomatériaux

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.

Une plate-forme technologique open source pour fabriquer des modèles de culture 3D basés sur l’hydrogel de manière automatisée et standardisée

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.

Isolement et culture de cellules épithéliales gingivales humaines primaires à l’aide de 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 ...

Génération évolutive de sphéroïdes cellulaires uniformes à l’aide d’un dispositif d’assemblage acoustique 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.

Pleins feux sur l’auteur : Développement d’une méthode d’assemblage acoustique sans échafaudage pour la culture de sphéroïdes cellulaires 3D de haute qualité 

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.

Dispositif d’assemblage acoustique tridimensionnel pour la fabrication en série de sphéroïdes cellulaires

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