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.
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.
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.
Her har vi vist anvendeligheden af menneskelig organotypisk bronchiale og nasale væv modeller til at vurdere virkningen af gentagen eksponering CS. Som et alternativ til dyreforsøg, blev en række eksponeringssystemer udviklet til toksikologiske vurderinger af aerosol eksponering in vitro (f.eks., Vitrocell, Cultex, Alice, etc.). Disse eksponering moduler kan også anvendes til en toksikologisk vurdering af luftbårne forurenende stoffer, luftbårne partikler, nanopartikler, etc. I denne undersøgelse anvendte vi Vitrocell system, der kan rumme op til 48 forskellige prøver samtidigt, giver mulighed for større skala eksperimenter og lavere variabilitet mellem behandlinger. For hver aerosol eksponering in vitro, risikoen for forurening vævskultur fortsat en stor risiko, hvis afbødning kræver en omhyggelig håndtering af vævskulturer hele eksperimenterne.
Måling partikelaflejring i realtid på mikrovægten under eXposure eksperiment gør det muligt at overvåge, at CS doser genereret fra eksponeringssystemet er i overensstemmelse med forventningerne. For at sikre nøjagtigheden af den målte partikelaflejring, oprettelse online måling korrekt, før eksponeringen er critial fx opsætning af skalaen til 0. Derudover grund af forskellen mellem de områder af mikrovægt (cm 2) og vævskultur insert (0,33 cm2), vi justeret den endelige beregning til området af kulturen insertet: kun 33% af aflejringen på mikrovægten afspejler den faktiske aflejring i kultur indsatsen.
Måling af TEER at fastslå stram-junction barriere funktionalitet og at vurdere afbrydelse af epitellaget er en forholdsvis nem procedure at gennemføre, som vi rapporteret her. Fordi de bronchiale og nasale væv modeller indeholde slim-producerende indflettede slimceller, skal imidlertid apikal vask skal udføres før TEER measurement. Den apikale vask er kritisk, fordi tilstedeværelsen af slim lag og variabiliteten af dens tykkelse dåse partiskhed måling af TEER, interferring med virkningerne af CS eksponering. Dette begreb er i overensstemmelse med, hvad der blev rapporteret af Hilgendorf og kollegaer, hvor permeabiliteten af Caco-2-celler blev berørt af co-dyrkning med slim-producerende bæger cellelinje HT29 23. Den slim skal vaskes før TEER måling fordi apikal vask lige før eksponeringen kan forstyrre de vævsreaktioner på CS. Derfor er målingen foretaget tre dage før eksponering, og ikke lige før eksponeringen.
Vi viste, at der kan måles CYP1A1 / CYP1B1 aktivitet fra de organotypiske kultur modeller efter CS eksponering selvom aktiviteten blev kun let øget med CS. Dette svagt signal kan forstærkes af en længere inkubation af CYP1A1 / 1B1 substrat (dvs.., Luciferin-CEE), for eksempel til24 timer (data ikke vist). En af de begrænsninger af CYP aktivitet måling i det foreliggende arbejde er fraværet af normalisering til CYP proteinniveau eller celletallet, som kan tages i betragtning ved fremtidige undersøgelser for at sikre, at ændringen på enzymaktiviteten ikke påvirkes ved ændring af enten protein eller det celletallet.
Vi rapporterede, at CS eksponering hæmmede ciliære slå i både nasale og bronkiale vævsmodeller. Lignende observationer blev gjort i forskellige pattedyr og ikke-pattedyr modeller 12. For ciliær slå måling sikre, at væv håndteres og behandles på en lignende måde er kritisk, for eksempel hvis medium ændring iværksættes, bør det anvendes til alle prøver. Sutto og kolleger rapporterede, at pH påvirkede pattedyr ciliær slagfrekvens 24. Således når man sammenligner slå frekvenser mellem celler behandlet med forskellige compunds / blandinger, pHjustering bør overvejes at minimere variabilitet af ciliære slå måling. Desuden temperatur, ved hvilken målingen udføres, er også kritisk, da hyppigheden af ciliære bankende falder med faldende temperatur. For at minimere variabilitet på grund af disse ændringer, en etape-top inkubator, udstyrede med temperaturen, CO 2 og fugtighed kontrol blev anvendt i denne undersøgelse. På trods af dette, vi observeret, at det ciliære slå frekvenser i de bronchiale væv efter CS eksponering var meget variabel (dvs.., Stige i et indlæg og falde i anden indsats), hvilket tyder på, at den ciliære bankende er meget forstyrret lige efter eksponering. I modsætning hertil observerede vi fraværet af målelige ciliær beating frekvenser i den nasale væv efter CS eksponering, hvilket tyder på, at reaktionen af den nasale væv er mere følsom og konsekvent. Dette er i overensstemmelse med en tidligere publikation viser, at den nasale væv har en lavere kapacitet til at afgifte somsammenlignet med bronkie 25.
Endelig viste vi, at genekspression profilering fra organotypisk bronchiale og nasale væv modeller udsat for CS kunne påvise en effekt af CS på xenobiotisk stofskifte. Interessant, den observerede ændring i xenobiotisk metabolisme i organotypisk bronchiale og nasal in vitro-modeller udsat for CS ligne in vivo-situationen hos rygere som diskuteret mere detaljeret i en tidligere publikation 15. For genekspression analyser, ved hjælp af robot instrument laver en high-throughput analyse mulig. Desuden automatisk robot håndtering øger yderligere sammenhæng og nøjagtighed genekspression resultater. Ikke desto mindre, hurtig samling af vævsprøverne var kritisk for at undgå RNA-nedbrydning under RNA-ekstraktion. RNA forarbejdning og transkriptom fremgangsmåder beskrevet her kan også anvendes til in vivo vævsprøver.
The authors have nothing to disclose.
Forfatterne vil gerne takke Maurice Smith og Marja Talikka for deres gennemgang af manuskriptet.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
MucilAir-human fibroblasts-bronchia | Epithelix Sárl, Geneva, Switzerland | http://www.epithelix.com/content/view/122/19/lang,en/ | |
MucilAir Culture Medium | Epithelix Sárl, Geneva, Switzerland | http://www.epithelix.com/content/view/84/16/lang,en/ | |
VITROCELL | VITROCELL systems GmbH, Waldkirch, Germany | http://www.vitrocell.com/index.php?Nav_Nummer=2&R= | |
3R4F reference cigarette | University of Kentucky | http://www2.ca.uky.edu/refcig/ | |
30-port carousel smoking machine SM2000 | Philip Morris, Int. | ||
CiliaMetrix camera and software | La Haute École de Gestion (HESGE), Geneva, Switzlerland | ||
Leica DMIL microscope | Leica, Heerbrugg, Switzerland | ||
LED light source | Titan Tool Supplies, Buffalo, NY | ||
Chopstick Electrode STX-2 | World Precision Instruments | http://www.wpiinc.com/products/physiology/stx2-chopstick-electrode-set-for-evom2/ | |
EVOMXTM Epithelial Voltohmmeter | World Precision Instruments | http://www.wpiinc.com/products/physiology/evom2-evom2-epithelial-voltohmmeter-for-teer/ | |
Luciferin Detection Reagent | |||
MagNA Lyser Instrument | Roche | http://www.roche.com/products/product-details.htm?region=us&type=product&id=66 | |
chloroform | Sigma-Aldrich | http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en®ion= | |
QIAcube | Qiagen | 9001882 | |
NanoDrop | Thermo Scientific | http://www.nanodrop.com/ | |
Agilent 2100 Bioanalyzer | Agilent | http://www.genomics.agilent.com/en/Bioanalyzer-System/2100-Bioanalyzer-Instruments/?cid=AG-PT-106 | |
Affymetrix GeneChip High throughput 3’IVT Express Kit | Affymetrix | http://www.affymetrix.com/catalog/prod370001/AFFY/High-Throughput-(HT)-Whole-Transcript-(WT)-Kit | |
Scanner 3000 7G | Affymetrix | http://www.affymetrix.com/catalog/131503/AFFY/Scanner-3000-7G |