Human organotypic buccal and small airway epithelial cultures

Organotypic human buccal epithelial (EpiOral™, MatTek Corp., Ashland, MA, USA) and small airway epithelial (SmallAir™, Epithelix. Geneva, Switzerland) cultures were used; each was reconstituted from the primary cells of a single donor (Table 1). A single donor was used to reduce the influence of donor-to-donor variability and hence to increase the statistical power to identify potential exposure effects, although the use of cells from a single donor can only indicate a donor-specific response. The cells were grown in Transwell® inserts (with a 6.5-mm diameter, and a 0.4-μm pore size) and maintained in 24-well culture plates at the ALI at 37°C (5% CO 2 , 90% humidity) in their respective culture media (0.7 mL/well) according to the manufacturers’ instructions.

Table 1 Human buccal and small airway culture models Full size table

The cultures were fully differentiated and acclimatized in the incubator before exposure. After exposure, the medium was not changed until it was collected for various endpoint measurements.

Reference cigarette smoke and electronic cigarette aerosols

Mainstream CS was generated from 3R4F reference cigarettes, purchased from the University of Kentucky, Lexington, KY, USA [27]. Test aerosols were generated from three different EL prototype formulations (Table 2), provided by Altria Client Services (ALCS) LLC, Richmond, VA, USA using MarkTen® EC devices (ALCS), Fig. 1.

Table 2 Electronic cigarette liquid formulations Full size table

Fig. 1 MarkTen® devices. A cartridge of MarkTen® contains 0.9 g of e-liquid. The puff activate product generates the aerosol via a 3.5 Ω (3.9 W) heater coil Full size image

Exposure setup

The study consisted of three experimental phases using buccal cultures and three phases using small airway cultures (Fig. 2). In each phase, three independent smoke and aerosol generations were performed (paired with their corresponding air-exposure controls), totaling nine independent generations (i.e., data points). For each phase, a new batch of cultures was used; exposures were conducted separately for each culture type. Various endpoints were assessed following exposures (Fig. 2).

Fig. 2 Experimental design and biological endpoints. Human organotypic buccal and small airway cultures were exposed in an in vitro aerosol exposure system (the Vitrocell® 24/48 exposure systems is illustrated). Three experimental phases, each including three independent aerosol/smoke generations, were performed using new batches of buccal and small airway cultures, totaling nine repetitions per culture type. Various endpoints were measured at specific time points before and after exposure, from each smoke/aerosol generation. PBS, phosphate-buffered saline Full size image

Two independent exposure systems (Vitrocell® 24/48; Vitrocell Systems GmbH, Waldkirch, Germany) were used: one for 3R4F reference CS, and the other for EC aerosol exposures. The Vitrocell® 24/48 exposure system is equipped with a Dilution/Distribution Module, into which fresh air can be added to dilute any aerosol serially in various rows (Fig. 2). During exposure, smoke or aerosol is passed through the Dilution/Distribution Module and distributed into the Cultivation Base Module via port ejectors (trumpets) by negative pressure [28]. The culture models were placed in the Cultivation Base Module and exposed to smoke or aerosol at their apical sides.

The aerosol generation from the EC aerosols was performed using single-programmable syringe pumps, which were connected to one dedicated Vitrocell® 24/48 exposure system. For each EL formulation, two MarkTen® devices were connected to one pump; a pinch valve—installed between the devices and the pump—alternated the activation of the two devices and resulted in the generation of one puff every 15 s (a total of 4 puffs/min was generated). Two devices (of the same formulation) were used simultaneously, in an alternate manner: a total of two devices was used during the 28-min exposure [one of the device was pre-puffed (57–112th puff were used), and the other was not (1st–56th puff were used)]. New devices were used for each aerosol generation (Fig. 2). Before being used for the experiment, the ECs were stored at room temperature in their original packaging.

The CS generation from the 3R4F cigarettes were performed using a 30-port carousel smoking machine (SM2000; Philip Morris, International), which was connected to another dedicated Vitrocell® 24/48 exposure system. A total of 4 puffs/min were taken consecutively from two 3R4F cigarettes placed in the 30-port carousel (puff frequency was every 15 s). For the 28-min exposure, a total of ten 3R4F cigarettes were smoked. The 3R4F cigarettes were stored in their original packaging at 5 ± 3°C with uncontrolled humidity conditions. Before being used for the experiment, 3R4F cigarettes were conditioned for between 2–10 days under controlled conditions at 22 ± 1 °C and a relative humidity of 60 ± 3% according to ISO guideline 3402 [29].

Smoke and aerosol generation was conducted according to the parameters given in Table 3.

Table 3 Smoke and aerosol generation parameters Full size table

For the current study, a set of organotypic cultures was exposed to various concentrations of 3R4F CS and to air, simultaneously, in one exposure system (Table 4). For 3R4F CS, cultures were exposed for 28 min (112 puffs) at various smoke concentrations (diluted in air). The concentrations of 3R4F CS were selected based on the sensitivity of human organotypic cultures observed in previous studies [31,32,33,34], from which we detected a considerable range of biological effects from gene expression to morphology. Another set of cultures was exposed to undiluted test mix, base, and carrier EC aerosols and to air, simultaneously, in another exposure system (Table 4).

Table 4 Smoke and aerosol exposure doses Full size table

Analysis of particle size distribution

The particle size distribution of the test mix, base, and carrier aerosols, as well as that of 3R4F CS, was measured upstream (before going into the Vitrocell® 24/48 exposure system) using the Aerodynamic Particle Sizer® (APS™, model 3321; TSI Incorporated, Shoreview, MN, USA), which was connected directly to the outlet of the single-programmable syringe pumps. This closed connection was established using a 1-m conductive tube with a 1-cm inner diameter. A T-junction (opens to the surrounding environment) was installed upstream of the APS™ to avoid buildup of negative pressure inside the connection that was expected as a consequence of the spectrometer-generated flow (at a volume-flow-rate of 5 L/min). The aerosol was supplied actively (by the action of the syringe pumps); therefore, the APS™ extracted only the volume of surrounding air necessary to compensate for the difference between the aerosol volume-flow-rate and the volume-flow-rate generated by the instrument; this meant that the complete aerosol volume was subjected to analysis. The particle concentrations in the 3R4F CS or EC aerosols provided by the pump were expected to be outside the working range of the APS™; therefore, a 100-fold dilution was applied by installing the 3302A Aerosol Diluter (TSI Incorporated) upstream of the APS™.

Analysis of nicotine in phosphate-buffered saline

Concentrations of the deposited nicotine in the exposure chamber were measured in the exposed PBS, which did not contain MgCl 2 or CaCl 2 (Sigma-Aldrich, St. Louis, MO, USA; Ref. D8357). One hundred microliters of PBS-filled steel inserts were located in the Base Module of the Vitrocell® 24/48 exposure system and exposed together with the buccal or small airway epithelial cultures, in every exposure experiment. Concentrations of nicotine were measured using liquid chromatography tandem-mass spectrometry.

Analysis of carbonyls in phosphate-buffered saline

The entire row of the Base Module of the Vitrocell® 24/48 exposure system was filled with PBS and exposed together with the epithelial cultures, in every exposure experiment. Before exposure, each row in the Cultivation Base Module of the Vitrocell® 24/48 exposure system was filled with 18.5 mL PBS. Following exposure, an aliquot of 1.2 mL PBS-exposed sample (per row) was collected and subjected to high-performance liquid chromatography coupled with tandem-mass spectrometry analysis, as previously reported [28].

Histology

Histological samples were obtained only from cultures harvested 48 h post-exposure, as conducted in our previous studies [35, 36] showing that morphological alterations would occur at later time points after molecular changes took place [37]. The processing of the organotypic cultures followed a previously published protocol [32]. Briefly, cultures were fixed for 2 h in freshly prepared 4% paraformaldehyde, and then removed from the insert for paraffin embedding using the tissue processor Leica ASP300S (Leica Biosystems Nussloch GmbH, Nussloch, Germany). Sections of 5-μm thickness were obtained and mounted on glass slides, which were subsequently stained with hematoxylin (Merck Millipore, Billerica, MA, USA), eosin (Sigma-Aldrich), and Alcian blue (Sigma-Aldrich). Digital microscopic images were generated using the slide scanner Hamamatsu NanoZoomer 2.0 (Hamamatsu Photonics, K.K., Hamamatsu, Japan). Histological assessment was conducted by a trained independent certified pathologist (Unilabs Independent Histopathology Services, London, UK). The specification of the various histopathological findings that were assessed is given in Supplementary Table 1.

Measurement of ciliary beating frequency

CBF measurement was conducted in small airway cultures only (not applicable for the nonciliated buccal cultures) using the Sisson Ammons Video Analysis system (Ammons Engineering, Clio, MI USA). Briefly, the ciliary beating videos were recorded using a video camera (Basler acA1300–200 µm; Basler AG, Ahrensburg, Germany) using a 4 × magnification (Leica DMi8 light microscope; Leica Microsystems, Heerbrugg, Switzerland) and following a set of parameters: a frame rate of 100 frames per second; a frame resolution of 640 by 480 pixels; a total number of 512 frames; and an 8-bit greyscale precision (256 levels of intensity). Ciliary beating of the cultures, on the field of interest selected from the center of each small airway culture, was measured before the exposure, immediately after exposure (0 h post-exposure), and 24 h and 48 h post-exposure. For the measurement, small airway cultures were transferred to a stage-top incubator (CU-501; Live Cell Instruments, Seoul, Korea). First, the data were processed by pixel, for which subsets of the pixels were processed individually. Their samplings were performed at regular steps, 1–8 on both directions (width and height), achieving an overall sampling ratio of 1 to 64. Then, the pixel-wise spectrum analysis was performed by: (1) centering the signal; (2) subtracting the mean of the pixel intensity over time (the 512 frames); (3) performing a fast Fourier transform on this signal, to estimate the periodogram power spectral density of this signal in the range from 0 to 50 Hz; and finally (4) smoothing the spectral density using penalized B-splines. Second, the data were processed by movies according to the following parameters: (1) the median spectral density was computed over the processed pixels by movies; (2) the median spectral density was smoothed using penalized B-splines; (3) the dominant frequency was determined as the frequency related to the highest spectral power in the range of expected cilia beating frequencies (2.5–25 Hz; the lower end, 2.5 Hz, was used to exclude high spectral power detected below 2.5 Hz, which was likely associated with movement of mucus above the culture that was visible in the video recording, while the upper end, 25 Hz, was used to exclude high-frequency noises often detected in severely damaged culture). The width of the peak containing the dominant frequency was taken as the frequency range of which the spectral power decreased monotonically from the peak; and (4) the weighted frequency was determined as the weighted mean of the peak frequencies (weighting the frequencies by their spectral power).

Measurement of secreted inflammatory mediators

Multi-analyte profiling of inflammatory mediators secreted into the basolateral medium of cultures was performed using commercially available Milliplex panels (Merck Millipore) with Luminex® xMAP® Technology (Luminex, Austin, TX, USA)-based analysis according to the manufacturer’s instructions. Briefly, 25 µL of diluted and non-diluted sample was used for each detection and the analysis was run on the FLEXMAP 3D® platform, equipped with xPONENT® software version 4.2 (Luminex). Data were presented as Median Fluorescent Intensity using a five-parameter logistic or spline curve-fitting method to calculate the analyte concentrations in the basolateral medium samples. The following analytes (inflammatory mediators) were measured: chemokine [C–C motif] ligand (CCL) 20; chemokine [C–X–C motif] ligand (CXCL) 1 (also known as GRO alpha), CXCL-8 (also known as interleukin [IL]-8) and IL-6; tumor necrosis factor alpha (TNFα); soluble intercellular adhesion molecule (sICAM) 1; matrix metalloproteinase (MMP)-1 and MMP-9; tissue inhibitor of metalloproteinase (TIMP) 1, and vascular endothelial growth factor (VEGF) alpha. The data were log-transformed, and the geometric means were used to calculate the fold-changes of the mediators (exposed vs. air-exposed samples).

Statistical analyzes

Basic descriptive statistical measures, such as mean, median, and standard deviation for all the investigated endpoints were computed (except for the analysis of mRNA data, see “Analysis of mRNA data” section). CS or EC aerosol-exposed groups were compared with the air-exposed controls using paired t test (paired within the same exposure run). The analyzes were performed in R-3.2.2 or R3.1.2, but the analyzes on ciliary beating was performed using SAS 9.2. Datasets, further detail on the protocols, and additional data visualizations are available on the INTERVALS platform at https://www.intervals.science/studies/#/MarkTen_Organotypic_PMI-Altria.

RNA isolation and array analyzes

Total RNA was isolated from the epithelial cultures using a previously published method [32, 34]. Briefly, for the mRNA array, 100 ng of total RNA was reverse transcribed to cDNA using an Affymetrix® HT 3′-IVT PLUS kit (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was labeled and amplified to complementary RNA (cRNA). The fragmented and labeled cRNA was hybridized to a GeneChip® Human Genome U133 Plus 2.0 Array (Thermo Fisher Scientific) in a 645 GeneChip® Hybridization Oven (Thermo Fisher Scientific) according to the manufacturer’s instructions. Arrays were rinsed and stained on a GeneChip® FS450 DX Fluidics Station (Thermo Fisher Scientific) using the Affymetrix® GeneChip® Command Console® Software (AGCC software v-3.2, protocol FS450_0001).

Finally, the arrays were scanned using a GeneChip® Scanner 3000 7G (Thermo Fisher Scientific). Raw images from the scanner were saved as DAT files. The AGCC software automatically gridded each DAT file image and extracted probe cell intensities into an Affymetrix CEL file.

Analysis of mRNA data

A model was fitted using limma R package [38] to estimate the treatment effect (for each experimental factor combination item, concentration and post-exposure duration) by including the covariate exposure run as a blocking variable to account for the pairing during an exposure run (exposed vs. air-exposed control samples). The p-values for each computed effect were adjusted across genes using the Benjamini–Hochberg false discovery rate (FDR) method [39]. Differentially expressed genes (DEGs) were defined as a set of genes whose FDR was < 0.05. The mRNA array datasets can be accessed in the Arrays Express repository (ID: E-MTAB-7577).

Processing raw CEL files from the mRNA microarray analyzes

The raw CEL files were background corrected, normalized, and summarized using the frozen-robust multi-array analysis [40]. Background correction and quantile normalization were used to generate microarray expression values from all arrays passing quality controls and were performed using the custom chip definition files environment HGU133Plus2_Hs_ENTREZG v16.0 [41], as previously described in greater detail [31, 33, 36].

Network-based enrichment analysis of transcriptomic data

Quantitative assessment of the transcriptomic data was conducted using a network enrichment approach and network perturbation amplitude (NPA) algorithm, described in greater detail in a previous publication [42]. Briefly, the methodology aims to contextualize transcriptome profiles (treated vs control, or aerosol/smoke-exposed vs air-exposed control samples) and quantify the biological impact of exposure by combining alterations in gene expression into differential network-node values, i.e., one value for each node of a causal network model [43]. Relevant network models used for the analysis in this study are listed in Supplementary Table 2. The selection of the network models was based on the relevancy to respiratory epithelium biology.

The NPA method uses transcriptome data without a fold-change or p value cut-off. The differential node values were determined by fitting procedures inferring the values that best satisfy the directionality of the causal relationships contained in the network model (e.g., positive or negative signs). NPA scores carried a confidence interval accounting for experimental variation, and the associated p values were computed. In addition, companion statistics were derived to permute the network structure, and the gene expression profiles were derived to inform the specificity of the NPA score to the biology described in the network models. The results from those permutation statistics were reported as *O and K* if their p values fell below the threshold of significance (0.05). A network was considered significantly affected by exposure if the three values (the p value for experimental variation, *O, and K*) were below 0.05 [42]. Network subgraphs (termed “NPA modules”), which are rich in leading nodes (i.e., nodes contributing the most to the NPA score of a network), were extracted by finding a maximum score of the connecting subgraph, using the sum of the leading node contributions as scores.

A system-wide metric for biological impact, the biological impact factor (BIF) [44, 45] summarized the impacts of the exposure on the cellular system into a single (absolute) number, thus enabling a simple and high-level evaluation of the treatment effects across multiple time points. Calculating the BIF required the collection of all applicable hierarchically structured network models (Supplementary Table 2), and involved aggregating the NPA score of the individual networks.