A review of in vitro cigarette smoke exposure systems

David Thorne; Jason Adamson

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Experimental and Toxicologic Pathology

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Review

A review of in vitro cigarette smoke exposure systems

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David Thorne *, Jason Adamson

British American Tobacco, Group R&D, Southampton SO15 8TL, UK

ARTICLE INFO

Article history: Received 8 February 2013 Accepted 13 June 2013

Keywords: In vitro Whole smoke Exposure systems Tobacco smoke Dosimetry Borgwaldt Burghart CULTEX® Vitrocell®

ABSTRACT

In vitro test methods may be vital in understanding tobacco smoke, the main toxicants responsible for adverse health effects, and elucidating disease mechanisms. There is a variety of 'whole smoke' exposure systems available for the generation, dilution and delivery of tobacco smoke in vitro; these systems can be procured commercially from well-known suppliers or can be bespoke set-ups. These exposure technologies aim to ensure that there are limited changes in the tobacco smoke aerosol from generation to exposure. As the smoke aerosol is freshly generated, interactions in the smoke fractions are captured in any subsequent in vitro analysis. Of the commercially available systems, some have been characterised more than others in terms of published scientific literature and developed biological endpoints. Others are relatively new to the scientific field and are still establishing their presence. In addition, bespoke systems are widely used and offer a more flexible approach to the challenges of tobacco smoke exposure.

In this review, the authors present a summary of the major tobacco smoke exposure systems available and critically review their function, set-up and application for in vitro exposure scenarios. All whole smoke exposure systems have benefits and limitations, often making it difficult to make comparisons between set-ups and the data obtained from such diverse systems. This is where exposure and dose measurements can add value and may be able to provide a platform on which comparisons can be made. The measurement of smoke dose, as an emerging field of research, is therefore also discussed and how it may provide valuable and additional data to support existing whole smoke exposure set-ups and aid validation efforts.

Contents

1. Introduction..........................................................................................................................................1184

2. Whole smoke exposure systems.....................................................................................................................1184

2.1. Bespoke systems..............................................................................................................................1185

2.2. Borgwaldt.....................................................................................................................................1186

2.3. Burghart......................................................................................................................................1187

2.4. Vitrocell® Systems............................................................................................................................1187

2.5. CULTEX®......................................................................................................................................1188

3. Dosimetry for smoke exposure......................................................................................................................1189

3.1. Deposition assessment using fluorometric analysis.........................................................................................1189

3.2. Light scattering photometers................................................................................................................1189

3.3. Quartz crystal microbalance.................................................................................................................1190

3.4. Vapour phase.................................................................................................................................1190

4. Discussion ............................................................................................................................................ 1191

Abbreviations: ALI, air-liquid interface; BAT, British American Tobacco; COPD, chronic obstructive pulmonary disease; CORESTA, Cooperation Centre for Scientific Research Relative to Tobacco; CSE, cigarette smoke extract; CuO, copper oxide nanoparticles; DMSO, dimethyl sulphoxide; DNPH, dinitrophenylhydrazine; HPLC, high performance liquid chromatography; 1ARC, International Agency for Research on Cancer; MAPK, mitogen-activated protein kinases; MSB-01, Burghart Mimic Smoker MSB-01; PAH, polycyclic aromatic hydrocarbons; PBS, phosphate buffered saline; QCM, quartz crystal microbalance; RFS, radial flow system; RM20S, Borgwaldt RM20S Smoking Machine; TSNA, tobacco specific nitrosamines; VC 10, Vitrocell VC 10 Smoking Robot. * Corresponding author. Tel.: +44 02380 588088; fax: +44 02380 588856. E-mail address: DavidThorne@bat.com (D. Thorne).

5. Conclusions..........................................................................................................................................1191

Declaration of interest.................................................................................................................................1191

Authors contributions..................................................................................................................................1191

Acknowledgements..................................................................................................................................1191

References...........................................................................................................................................1192

1. Introduction

In order to understand the various physiological and phys-icochemical processes associated with tobacco consumption, especially disease associations, we must first understand the complex dynamics of tobacco smoke, which may allow the precursors and mechanisms responsible for adverse health effects to be ascertained. The approximate composition of mainstream tobacco smoke is relatively well defined, due to decades of research and advances in analytical techniques (Borgerding and Klus, 2005; Liu et al., 2011; Talhout et al., 2011). Tobacco smoke is a complex and dynamic aerosol consisting of thousands of chemicals, the most recent estimate is 5600 individual smoke components (Perfetti and Rodgman, 2011) of which approximately 158 have toxicological properties - termed tobacco smoke toxicants (Fowles and Dybing, 2003). Distributed between the particulate and gaseous fractions and sometimes present in both are chemicals known to be associated with various smoking related diseases. For example; the aldehydes (formaldehyde, acrolein, acetaldehyde) are found in the gaseous phase (Baker, 2006) of cigarette smoke and are associated with chronic obstructive pulmonary disease (COPD) and lung toxicity (1ARC, 1985; Barnes, 2003; 1ARC, 2006). Polycyclic aromatic hydrocarbons (PAHs), tobacco specific nitrosamines (TSNAs), arsenic, cadmium and chromium are found in the particulate phase of cigarette smoke and can be linked with various cancers (1ARC, 1990,1993; Hoffmann and Hoffmann, 1997; Levitz et al., 2004).

Tobacco smoke assessment in vitro has traditionally focused on the particulate phase captured on a Cambridge filter pad and eluted in DMSO (Crooks et al., 2013) or bubbled through cell culture media or PBS (Andreoli et al., 2003). Cell cultures are then exposed under submerged conditions to the particulate phase. Unfortunately, particulate based exposure scenarios do not take into account the vapour phase of cigarette smoke, or the associated interactions between the particulate and vapour phases. Submerged culture conditions and particulate-based exposures do not represent physiologically that of mainstream tobacco smoke exposure in the human lung. Furthermore, separating smoke fractions in this way could lead to alterations and chemical changes that may not be representative of the whole smoke aerosol. In order to address these challenges, whole smoke exposure systems have been developed. Whole smoke exposure systems offer many technical challenges, but represent a more physiologically relevant test system that captures the full interactions of both the particulate and vapour phase together (Fukano et al., 2004). An additional advantage of these systems is that a multitude of different cell cultures can be exposed at the air-liquid interface (ALI) to whole smoke, better simulating human exposure.

There are a diverse range of whole smoke exposure systems available ranging from commercial set-ups (Aufderheide and Mohr, 1999; Phillips et al.,2005; Scian et al., 2009a; Okuwa et al.,2010)to bespoke in-house designed and developed exposure systems (St-Laurent et al., 2009; Muller et al., 2011; Zhang et al., 2011; Gualerzi et al., 2012). Irrespective of origin, these systems generally have in common two main components: (1) a smoking machine, which generates, dilutes and delivers cigarette smoke; (2) an exposure chamber which houses the associated biological system often (but not always) at the ALI. The amount of smoke delivered within these in vitro exposure systems can be presented in many ways, often

dependent upon the machine used. Delivered smoke can be presented either as a ratio of smoke to air, a flow rate of mixing air applied to the smoke dilutor, as a percentage of smoke, as smoke fraction, or as cigarette puff number. As there is no consensus on how in vitro smoke data should be presented, this makes comparisons of research difficult. What is more relevant and is becoming increasingly important in the field of in vitro whole smoke assessment is dosimetry: i.e. how to quantify the smoke dose to which the cells are directly exposed. As there is not a consistent approach to whole smoke exposure, with a variety of systems and laboratory set-ups being used, dose tools could play an important role and bridge the gap between technologies; not only in the measurement of actual cellular dose but also in the characterisation and validation of these systems. Furthermore, utilising dose tools will add strength to the resulting in vitro data and potentially allow cross-platform comparisons to be made, where currently they cannot.

2. Whole smoke exposure systems

The development of whole smoke exposure systems has been driven by the fact that traditional smoke exposure techniques are based on the particulate phase of cigarette smoke and omit the vapour phase and semi-volatiles from any subsequent analysis. Whole smoke exposure systems offer the advantage that all phases of smoke can be analysed together or independently depending on the experimental set-up. This has allowed researchers to tailor their experiments to investigate both phases of tobacco smoke, yielding useful information. There are a variety of whole smoke systems available and the majority of these systems can also be used to deliver individual aerosols or other complex aerosol mixtures to cell cultures. Consequently these systems can have applications outside the tobacco industry, however limited information and fewer guidelines exist for the assessment of airborne chemicals in vitro (Costa, 2008; Bakand and Hayes, 2010). Examples of commercially available systems include (but are not limited to): the Borgwaldt RM20S (Borgwaldt KC, Hamburg, Germany); the Burghart MSB-01 (Burghart Tabaktechnik Wedel, Germany); and the Vitrocell® VC10(Vitrocell® Systems, Waldkirch, Germany). Commercially available exposure chambers or modules which are linked with these machines include those supplied by British American Tobacco (Curbridge Engineering, Southampton, UK); CULTEX® (CULTEX® Laboratories Hannover, Germany); and Vitrocell® (Vitrocell® Systems, Waldkirch, Germany). There are pronounced similarities between a number of CULTEX® and Vitrocell® exposure modules, and this is due to their shared inception. However, after seven years of scientific, technical and commercial cooperation (1999-2006) on the development of approaches to in vitro toxicity testing, the working team split and formed what is today known as the CULTEX® and Vitrocell® groups. This explains similarities and compatibilities between the two systems as well as the diversity in more recent developments. In addition to commercially available systems some examples of bespoke systems are also discussed.

A summary of the commercially available and published in vitro exposure smoking machines and exposure chambers can be found in Tables 1 and 2 respectively.

Table 1

Comparison of the technical specification of the 3 main commercially available smoking machines. All measurements made by authors (□) and information obtained from technical support and commercially available sources (*), unless otherwise referenced.

Smoking machine Borgwaldt RM20S Smoking Burghart MSB-01 Mimic Smoker Vitrocell VC10 Smoking Robot

Machine

Dimensions (L x D x H)

Footprint

Dilution system

Dilution range Throughput

Computer controller Smoking regime

Tubing length to exposure device Exposure chamber/module

Time taken from puff to exposure Air flow controller

2.4 m x 0.8 m x1.3mn Free standing (2 m2 footprint)0 Syringe based independent dilution system (capable of 8 dilutions) (Adamson et al., 2011) 1:2-1:4000 (smoke:air, v/v) (Kaur et al., 2010) 8 chambers with 3, 6, 8 inserts/chamber0

Integrated computer0 ISO and HCl*

~3.4mn

Predominantly the British American Tobacco Exposure chamber - manufactured by Curbridge Engineering ~15-24 s (depending on dilution)0 Integrated

0.75 m X 0.35 m x 0.48 m* Bench top (0.8 m2 footprint)* Syringe based independent dilution system (capable of 5 dilutions) (Scian et al., 2009a) 1:1-1:150 (smoke:air, v/v) (Scian et al., 2009a) 96 well format*

Integrated computer* ISO, HCI and multi-smoking regimes* -1 m*

Integrated multi-well 24 and 96 plates

-6 s (Scian et al., 2009a) Integrated

1.5 m X 0.8 m x 0.85 m° Bench top (1.2 m2 footprint)° Continuous flow dilution bar (capable of 4 dilutions) (Okuwa et al., 2010)

Airflow 0-12l/min and vacuum rate 5-200 ml/min° 4 modules with 3, 4 inserts/module; 96 or 24 well plate manifold°,* Requires PC°

ISO, HCI and bespoke smoking regimes (Li et al., 2012) ~1.4m°

Vitrocell® orCultex® exposure modules

Additional equipment required

2.1. Bespoke systems

Bespoke exposure set-ups tend to be utilised by those involved in fundamental tobacco smoke research. Although there is little room for comparison between commercially available and bespoke systems, these one-off-a-kind set-ups offer the benefit of lower cost, smaller footprint, reduced complexity and often ease of maintenance. There are a wide range of bespoke designs and setups ranging in sophistication and offer unique and often simple cigarette smoke generation, dilution and exposure characteristics. Here we explore a few examples of how bespoke systems can be used to generate meaningful biological data.

St-Laurent et al. (2009) described one such system for the ALI exposure of isolated rat bronchial epithelial cells to the mainstream smoke from two cigarettes. The exposure system consisted of a 'hermetic chamber with two ventilation holes' large enough to accommodate a cell culture plate, and installed with a small fan. Cells were exposed to smoke twice daily for three consecutive days and cell supernatants were obtained at baseline and day three for subsequent mediator (MCP-1, IL-10, VEGF) assessment by enzyme-linked immunosorbent assay. The ALI results were also compared to cells exposed to cigarette smoke extract (CSE) under a submerged exposure condition. Analysis of the ALI results demonstrated that MCP-1 release was inhibited compared to the CSE model, IL-10 production was reduced, and VEGF showed no difference in production

between the two models. The authors concluded that the CSE and ALI models modulated bronchial epithelial cell mediator production differently, demonstrating that the model used can influence the data obtained (St-Laurent et al., 2009). Additionally, this suggests that varying cellular responses are observed when using different tobacco smoke fractions, indicating that different smoke fractions have independent roles in mediating tissue responses.

In another example, an exposure chamber (Phillips et al., 2005) was paired with a simple in-house cigarette smoke generator to assess the effect of smoke on human (healthy smokers and individuals with COPD) brushed bronchial epithelial cells and their innate immune response to Moraxella catarrhalis infection (Zhang et al., 2011). One cigarette was combusted using a pump and smoke was drawn into a 1000 ml flask for a 10min exposure to cells housed at the ALI. The results demonstrated that tobacco smoke increased bacterial load but decreased prostaglandin E2 (PGE2) production; PGE2 having been shown to exert immunomodulatory functions. The authors commented that the results help to clarify the role of PGE2 in mucosal innate immunity of COPD patients (Zhang et al., 2011).

In a final example, a cigarette, syringe and a sterile flask were connected by tubes in a T-shape via a tap at the connection point (Gualerzi et al., 2012) to create an uncomplicated yet unique setup. The sterile flask contained keratinised oral mucosa explants from healthy non-smoking woman, in a semi-immersed condition

Table 2

Comparison of the technical specification of 4 different commercially available exposure chambers. All measurements made by authors (□) and information obtained from technical support and commercially available sources (*), unless otherwise referenced.

Exposure chamber Curbridge Engineering Cultex® exposure module Cultex® radial flow system Vitrocell modules

(British American Tobacco) (RFS) (PT-CF/Ames)

Approximate dimensions (D x W x H) 12 cm x 9cmn 10cm x 16cm x 13cm* 35 cm x 24 cm x 20 cm0 10cm x 16cm x 13cm^

Approximate weight 0.65 kgn 2.5kg* 11.5 kg0 4.5 kg^

Material Transparent Perspexn Polished stainless steel and Polished stainless steel and Polished stainless

glass* glass* steel/glass and aluminium*

Capacity 3 x 24 mm 0 insertsn 3 x 24 mm 0 inserts* 3 x 24 mm 0 inserts* 3 or 4 x 24 mm 0 inserts*

6 x 12 mm 0 insertsn 3 x 12 mm 0 inserts* 3 x 12 mm 0 inserts* 3or4 x12mm0 inserts*

8 x 6.5 mm 0 insertsn 3 x 35 mm 0 Petri dishes* 3 x 6.5 mm 0 inserts* 3 x 35 mm 0 Petri dishes*

3 x 30 mm 0 Petri dishesn Petri dishes*

1 x 85 mm 0 Petri dishn

Chamber smoke delivery Sedimentation, Brownian Guided via funnel-shaped Sedimentation, diffusion, Direct exposure technology

motion, gravitation* metal inlet tube (trumpet)* electrical forces and inertial impaction (Aufderheide et al., 2011) (trumpet)*

to better represent the physiological condition of the oral cavity. A single cigarette was smoked for 6 min by 'inhaling' through the syringe, turning the tap to exhale the smoke to the flask, then detachment of the flask to allow the outflow of smoke and air recirculation; the whole cycle being repeated until the cigarette was smoked. Post-exposure, biomarkers of intracellular adhesion were evaluated by histochemical and immunofluorescence analysis. The results obtained suggested that the first response to cigarette smoke came from the basal/suprabasal layers of the oral epithelium, with an overexpression of keratin protein (K14) as early as 3 h after smoke exposure. Furthermore, this set-up maintained the 3D arrangement of the human mucosa and allowed a quasi-replication of the inhalation/exhalation cycle (Gualerzi et al., 2012).

2.2. Borgwaldt

The Borgwaldt RM20S (Borgwaldt KC GmbH, Hamburg, Germany) is a rotary style, syringe smoking machine, capable of smoking up to eight cigarettes simultaneously (Fig. 1) without cross-cigarette contamination. The combination of smoking allows either eight different cigarettes to be assessed using one dilution or one cigarette type at eight dilutions. Thus the RM20S smoking machine can be used for both product assessment purposes and fundamental research. Smoke is generated via a syringe which draws a puff from the cigarette first, sequentially followed by a puff of filtered air to create the required dilution, expressed as a ratio of smoke in air (1:X, volume:volume). Larger dilutions require a serial dilution process. Diluted smoke is exhausted from the syringe at 0.8 l/min into the exposure chamber. Each syringe is attached to an individual exposure chamber which ensures that no cross-cigarette or cross-dilution contamination can occur. Dilution of smoke and cellular exposure can take anywhere between 12 and 24 s depending on dilution.

Although not supplied together, and designed to be interchangeable, the Borgwaldt RM20S has almost exclusively been paired with the British American Tobacco (BAT) exposure chamber manufactured by Curbridge Engineering (Southampton, UK) (Fig. 2). This specific combination has been documented through a series of in vitro development and machine characterisation papers (Phillips et al., 2005; Maunders et al., 2007; Thorne et al., 2009; Kaur et al., 2010; Adamson et al., 2011, 2013a).

Phillips et al. (2005) first investigated cellular responses in bronchial epithelial NC1-H292 cells exposed to cigarette smoke, using the RM20S and BAT exposure chamber. This study

Fig. 1. A Borgwaldt RM20S smoking machine with 8 syringes. (A) Cigarette smoke generator. (Bi) A syringe based dilution system with 4 syringes. An additional 4 syringe unit is available from Borgwaldt KC GmbH, Hamburg Germany (Bii). (C) Air flow controller. (D) Cell culture media maintained at 37 °C feeding exposure chambers with fresh cell culture medium and (E) a BAT exposure chamber housed at 37 °C, attached to the smoke diluter and culture media (modified from Adamson etal., 2011).

demonstrated cytotoxicity, particulate deposition, mRNA (MUC5AC) and protein expression (Interleukin 6, 8, GRO-a and matrix metalloproteinase 1) in response to tobacco smoke exposure. Maunders et al. (2007) followed up this work using primary human lung bronchial epithelial cells focusing on gene expression changes following smoke exposure using Affymetrix and microarray technology. The results were consistent with previously reported in vitro and in vivo studies on smoke toxicity, such as increased epithelial permeability, antioxidant responses and MAPK pathways (Hackett et al., 2011). Maunders et al. (2007) also concluded that the down regulated responses in cell adhesion may provide a possible mechanism for smoke induced permeability and have implications in the development of various tobacco smoke induced diseases.

Initial characterisation of the RM20S was conducted by Kaur et al. (2010) who compared two RM20S machines in different

Fig. 2. British American Tobacco's exposure chamber manufactured by Curbridge Engineering (Hampshire, UK) and a schematic cross-section (Thorne et al., 2009). The exposure chamber introduces the test aerosol through a single gas inlet which creates a passive, sedimentary exposure scenario. A symmetrical smoke distribution plate ensures uniform cellular exposure.

Fig. 3. Schematic cross-section of the Vitrocell® VC 10 Smoking Robot. (A) Smoking robot carousel and side stream chimney, where cigarettes are loaded and smoked. (B) Piston/syringe which draws and delivers ISO (35 ml) or HCI (55 ml) mainstream cigarette smoke. (C) Air jets add continuous diluting air perpendicular to the mainstream smoke in the range of 0.2-12l/min; rates are set and maintained by mass flow controllers. (D) Dilution, transit and delivery of whole smoke occurs in the dilution bar. (E) Isolated cell culture inserts are supported and exposed to diluted whole smoke at the ALI in a module which docks under the dilution system. (F) Negative pressure is applied to the module which draws the diluted smoke from the dilution bar, into the module via the 'trumpet' inlets and out of the module through the exhaust. Volumetric flow rates, applied via a vacuum per well can vary depending on exposure conditions from 5 to 200ml/min, however a consistent flow rate per well of 5 ml/min is normally observed. (G) Due to continuous diluting airflow, smoke remaining in the dilution system transits to exhaust away from the module (modified from Adamson et al., 2013b).

geographical locations, Canada and the UK. Using a hydrocarbon analyser, a 10% methane gas standard, syringe dilution and precision across the two locations were assessed. The results demonstrated that although there was syringe variability, both machines in different locations were similar in performance (<10% relative standard deviation). Further characterisation was conducted by Adamson et al. (2011) who described losses in the system equivalent to those reported for the Burghart MSB-01 (Scian et al., 2009a). By using an electromobility spectrometer (DMS500 Cambustion, UK) at various positions along the system (post-puff, pre-chamber and post-chamber) they were able to demonstrate ~48% loss of mainstream smoke before cellular exposure and ~16% deposition within the chamber.

One of the main limitations of the RM20S exposure system and associated chamber combination is that to date it has been almost exclusively used by British American Tobacco making direct comparisons between other studies difficult.

2.3. Burghart

The Burghart Mimic Smoker MSB-01 (Burghart Tabaktechnik, Wedel, Germany) differs from other commercially available smoke exposure systems in that it has an integrated multi-plate format, designed for high throughput in vitro experimentation. The exposure plate (96 microwell format) is integral to the exposure system and therefore offers a simplistic approach free from the complication of an associated independent exposure module. The MSB-01 smoking machine is designed with independent syringes which enable a range of cigarettes or doses to be assessed. The syringes have a dilution capability up to 1:150 (smoke:air, v:v). A smoke distribution plate or manifold provides a consistent exposure via two ports across the multiwell plate. Cigarette puff to exposure takes approximately 6 s.

Characterisation of the MSB-01 was conducted by Scian et al. (2009a,b) including particulate deposition in the multiwell plate, smoke loss measurement, particle size, and cellular viabilities. Deposition on the exposure microwell plate was determined by optical fluorescence (370 nm) of particulate matter dissolved in

DMSO, whilst smoke loss measurements were determined by par-ticulate capture at various points in the system. Bronchial epithelial BEAS-2B cells were used to assess cytotoxicity following smoke exposure. The information accumulated has resulted in a good understanding of how the MSB-01 performs with regards to smoke losses, deposition and dilution reproducibility. Furthermore, analysis of cigarette smoke chemistry within the system has helped in the understanding of how the machine delivers smoke and the effects of dilution on particle size and distribution. Reported loses in this system preceding exposure were similar to those identified for the Borgwaldt RM20S, between 40% and 50% (Adamson et al., 2011; Scian et al., 2009b).

Compared to other smoke exposure machines, the MSB-01 is a higher throughput option. However, one potential drawback of this technology is that cells are not supported at the ALI, which ultimately limits the MSB-01 for use in simplistic cellular studies. There are also set-up implications such as a smoke collection chamber and a 'mixing' bag which may artificially age smoke (Scian et al., 2009a).

2.4. Vitrocell® Systems

The Vitrocell® VC 10 Smoking Robot (Vitrocell® Systems GmbH, Waldkirch, Germany) is a rotary, single syringe, continuous diluting flow smoking machine (Fig. 3). The VC 10 Smoking Robot has a syringe which transfers mainstream cigarette smoke to an independent continuous flow dilution system comprising of four or five dilution bars. Air is added above and below the dilution bar, perpendicular to the stream of cigarette smoke and mixes creating a turbulent stream of diluted cigarette smoke. Smoke dilutions are created by increasing or decreasing the airflow. In addition, a sub-sample of smoke is drawn from the dilution bar into the module through negative pressure applied via a vacuum pump.

The VC 10 Smoking Robot is usually paired with the Vitrocell® or CULTEX® exposure modules as their designs are complementary (Nara et al., 2013). The exposure modules dock directly under the continuous flow dilution system. Inserts containing cells are exposed separately to diluted smoke from the dilution bar.

Smoke/aerosol in Smoke/aerosol out

Island - can be unscrewed Cells grown

and replaced with a QCM on porous

membranes

Fig. 4. Schematic cross-section of the 3-well mammalian Vitrocell® 6/4 CF Stainless module during exposure. (1) The module lid has specially designed (trumpet) inlets for optimal aerosol distribution and particle deposition. All smoke inlets dock to the smoke dilution bar during exposure. (2) Integrated with the lid is the aerosol outlet which is attached to a vacuum pump. (3) The medium is supplied individually for each of the 3 well compartments and does not transit between them. Fresh medium exchange can be performed on a continuous basis per well compartment using a precision medium pump. Constant temperature of the unit is assured by a regulated flow of temperature controlled waterthrough the module lid and base. The central islands can be unscrewed to allow the incorporation of the quartz crystal microbalance (QCM), for assessment of particulate deposition.

Furthermore, Vitrocell® have developed several exposure module adaptations. For example, there is a mammalian 3 well (Persoz et al., 2010) and 4 well exposure module for various Transwell® sizes (Fig. 4) and a 24 and 96 well culture plate manifold for higher throughput. In addition, Vitrocell® also supply a bacterial exposure module for the Ames assay. All exposure modules utilise the same 'trumpet' technology that is shared with CULTEX®, which directs the exposure aerosol onto the cells, facilitating a more active diffusion and deposition environment within the module.

Okuwa et al. (2010) demonstrated the use of the VC 10 to assess the induction of micronuclei in Chinese hamster lung following exposure to mainstream cigarette smoke using multiple smoking regimes. In addition, the authors compared the effects of whole smoke versus vapour phase. Interestingly, Okuwa and colleagues noted a difference in micronuclei induction for both smoking regimes and also differences between the particulate and vapour phases of mainstream cigarette smoke - indicating that both smoke fractions have an importance in micronuclei induction. Furthermore, the authors used photometer light-scattering technology as a real-time measurement tool to semi-quantify and monitor smoke delivery, ensuring robust and repeatable data.

Like other exposure systems, the VC 10 has its limitations. At present there are few scientific publications on the application of the VC 10 or on the characterisation of the exposure system itself. Furthermore, the VC 10 has many variables that can be altered to create the required exposure set-up, for example: the diluting airflow (l/min) and module sampling flow-rate (ml/min/well) can be adjusted independently. This flexibility, although a potential positive is currently a limitation as the manipulation of these variables has yet to be fully characterised. However, this is changing and a recent study has assessed these variables on particle deposition in the exposure module. Interestingly, increased module flow-rates (ml/min) had an inverse effect on particulate deposition within the exposure module (Adamson et al., 2013b).

2.5. CULTEX®

CULTEX® Laboratories (Hannover, Germany) offer solutions for in vitro toxicological analysis of airborne substances, such as gases, particles, volatile compounds and complex gas mixtures at the ALI. CULTEX® supply in vitro exposure modules which have been designed to be used with a variety of exposure systems and

aerosols, making them adaptable and applicable to a multitude of exposure scenarios. As a result, CULTEX® play an active role in research surrounding in vitro aerosol exposure and inhalation toxicology.

Over the last decade, the CULTEX® exposure modules have been used in studies to assess a multitude of complex aerosols, such as diesel exhaust, cigarette smoke, therapeutics, volatile compounds, particles and environmental pollutants (Aufderheide and Mohr, 1999, 2000, 2004; Ritter et al., 2001, 2003; Aufderheide and Gressmann, 2007; Aufderheide et al., 2011; Deschl et al., 2011). The diversity of these studies demonstrates the scale and potential application of the CULTEX® exposure modules.

Aufderheide and colleagues first introduced the concept of the CULTEX® exposure module in 1999 and 2000. In the first two publications the authors outlined the use of the CULTEX® exposure modules for the in vitro assessment of inhalable test compounds (particles, mineral fibres and wood dust) at the ALI. In 2001, Ritter et al., used test synthetic air, ozone (202-510 ppb) and nitrogen dioxide (75-1200 ppb) to characterise exposure conditions within the CULTEX® module, indicating that this module can support a variety of approaches in the field of environmental toxicology for airborne chemicals. Further characterisation of the CULTEX® module was carried out by Aufderheide et al. (2003) who assessed particulate deposition using a fluorescence spectrophotometer technique, viability and cellular glutathione levels following exposure to tobacco smoke.

In 2007, Olivera et al., published on epithelial tight junction permeability using a CULTEX® module, in response to mainstream cigarette smoke. Using this in vitro exposure system they deduced a possible mechanism for loss of tight junction stability seen in respiratory epithelium in smokers.

More recently, Deschl et al. (2011) used the CULTEX® module combined with therapeutic aerosols to assess biological effects. The authors demonstrated that this exposure system can be used to understand disease mechanisms and as a tool to assess therapeutic efficacies in vitro, thus potentially reducing the need for in vivo experimentation in the future. Additionally, the authors noted that the linear arrangement of the aerosol sampling points along the exposure module gave varying results within the module, which could be caused by potential a potential turbulent mixing inefficiency and the linear arrangement of the sampling ports form the dilution system. The authors also commented that future iterations of this

technology may eliminate any potential limitations and uncertainties (Deschl et al., 2011).

The latest development from CULTEX® is the radial flow system (RFS) which allows culture medium to be precisely controlled (Fig. 5). The radial arrangement of cell culture inserts around a central gas inlet provides homogenous distribution (Aufderheide et al., 2011). The RFS utilises 'trumpet' technology which facilitates delivery of the aerosol almost directly onto the cell monolayer. Using the RFS, Aufderheide et al. (2011) demonstrated cytotoxic responses in human bronchial epithelial 16HBE cells following exposure to cigarette smoke. Furthermore, the authors used the RFS for the assessment of revertant colonies using the Ames test (Salmonella typhimurium strains TA98 and TA100) following exposure to tobacco smoke. In a more recent study (Aufderheide et al., 2013) the RFS module demonstrated delivery of a uniform test aerosol to the three culture inserts. This information was in part obtained using computational fluid dynamics to acquire greater insight into the flow conditions within the RFS module. The RFS module fundamentally differs from the linear glass module in that culture inserts are arranged symmetrically around a single gas inlet, thus eliminating any concentration gradient observed potentially due to linear sampling points. In addition to delivery of a homogenous test article, the RFS module also allows for the incorporation of different cell culture inserts and petri-dishes for the Ames assay. Interestingly, the authors also showed a correlation between particle size and distribution against deposited mass for aerosolised copper oxide nanoparticles (CuO). For example, direct exposure of CuO showed increased cytotoxicity in A549 cells compared to micro-sized particles, suggesting that cells were exposed to a greater number of smaller sized particles per cm2 area (Aufderheide et al., 2013).

CULTEX® Laboratories supply a diverse range of exposure chambers for a variety of exposure scenarios and aerosol test compounds. CULTEX® Laboratories also offer a dust aerosol generating system, which is comprised of three independent components; a HyP-Hydraulic Press for preparing powder cakes from a variety of powdery materials, a dust generator (CULTEX® DG) which provides uniform concentrations of aerosolised dust, and finally, an integrated elutriator, which stores the dust aerosol, serves as a reservoir for generated particles and facilitates uniform aerosol delivery (Aufderheide et al., 2013).

3. Dosimetry for smoke exposure

As tobacco smoke is a physically and chemically complex mixture, it is important to understand exactly what components of smoke the cell cultures in smoke exposure systems are being exposed to. There are plenty of opportunities for the smoke aerosol to coagulate and deposit during transition through whole smoke exposure systems; in addition, deposition efficiencies differ between systems. Therefore, exposure concentrations set on these smoking machines will not always resemble that of the actual cellular dose.

Tobacco smoke has two phases which contribute in distinct ways to lung injury and cellular damage. It is therefore important from a dosimetry perspective to understand the characteristics and interactions of both of these phases. Forthe deposited fraction, concentration, particle size and mass can be measured with standard aerosol monitoring devices and has previously been documented (Adam et al., 2009; Hofmann et al., 2009). Additionally, several recent and comprehensive reviews in the area of aerosol science and biology have been conducted (Grass et al., 2010; Paur et al., 2011). In terms of in vitro exposure, especially for those systems relying on diffusion and/or sedimentation, particle deposition efficiency is relatively low, about 16% (Desantes et al., 2006). However,

recent technical developments have seen the use of electrostatic precipitation to increase experimentally determined overall deposition efficiencies under the influence of an alternating electrostatic field to 15-30% when applied to monodisperse polystyrene particles in the size range of 50-600 nm (Savi et al., 2008). This allows researchers to tailor their exposure system to maximise deposition efficiency (Bruijne et al., 2009). Electrostatic precipitation devices are commercially available from CULTEX® (EDD), which offers the added benefit that they can be used in conjunction and are compatible CULTEX® exposure modules. Using this system particle deposition efficiency especially for those particles not normally deposited through sedimentation or diffusion can be increased up to 95% (Aufderheide et al., 2013).

There are a number of physical, chemical and gravimetric methods for determining tobacco smoke dosimetry in vitro. Most techniques have limitations and there is no general consensus on the most appropriate approach to quantifying dose. Some techniques look at specific tobacco smoke markers such as solanesol in the particulate phase, or carbon monoxide in the gas phase, whilst others quantify smoke in larger groups/families of chemicals. The majority of dosimetry techniques and measurements are based on the particulate phase of cigarette smoke, mainly due to the challenges associated with measuring vapour phase markers. Attempts have been made to use more sophisticated techniques for vapour phase analysis. In 2011, Kaur et al., described a headspace stir bar-sorptive extraction GC-MS technique for the assessment of cigarette smoke vapour phase compounds (volatile and semi-volatile compounds) including; 2-methyl-1,3-butadiene, 3-methyl-2-butanone, benzene, 2,5-dimethylfuran, toluene, ethylbenzene, p-xylene, styrene, 1-methy-4-(methyl-ethylidene)cyclohexane and limonene during in vitro exposure. Such techniques may not allow for real-time analysis, but may provide answers as to the relationship between smoke constituents and dose. In addition these tools may also be applied in a cross-platform approach.

The development of dosimetry tools to measure the vapour phase of tobacco smoke in vitro is a massive challenge (Lin et al., 2012). Currently, there is a trend to measure the particulate phase of tobacco smoke within these exposure system. In the following sections, a variety of available dose tools for particulate and vapour are discussed in more detail.

3.1. Deposition assessment usingfluorometric analysis

One method for the quantification of particulate deposition is chemical spectrofluorometric analysis (Ritter et al., 2003). In brief, pre-wetted cell culture inserts housed within the exposure chamber are exposed to whole mainstream smoke. Deposited particulate material can be extracted using high performance liquid chro-matography (HPLC) grade methanol and agitation, and extracts analysed with HPLC and fluorescence detection with standard calibration curves (Adamson et al., 2011,2012). This method, although not real-time, offers a simplistic approach to quantifying dose and has been used in a variety of studies. The advantage of this wet-chemistry technique is it can be applied to any exposure system/exposure chamber format (small well plates up to Petri-dishes) and results should be relatively consistent and comparable across laboratories.

3.2. Light scattering photometers

Photometers are designed to perform in-line measurements of the particle droplet suspended in the gas (termed optical tar) via a light scattering optical sensor. They are capable of measuring optical density at very low flow rates without any losses to the particle mass. Photometer technology has been around for many

years and has been used in a variety of studies, both in vivo and in vitro (Bellmann et al., 2009; Okuwa et al., 2010). The technology we specifically refer to here is a condensed portable version that can be incorporated into any aerosol exposure system for the direct analysis of aerosol density without effecting the aerosol stream. Due to its size and versatility, photometer technology can offer many advantages to an in vitro exposure system. Okuwa et al. (2010) demonstrated this by publishing data using portable photometers (Vitrocell® Systems, Waldkirch, Germany) in-line as a real-time monitoring tool. In this system the authors were able to monitor and quantify the amount of cigarette smoke particulate matter entering the chamber during exposure in real-time. This information is invaluable in ensuring the exposure conditions are both consistent and reproducible. Photometer technology does have its limitations. One such limitation is that these photometers have to be precisely calibrated against a known particulate mass; furthermore, in-line particulate measurements do not always resemble (although may give an indication of) deposited mass at the ALI.

3.3. Quartz crystal microbalance

Quartz crystal microbalance (QCM) technology has also been around for many years and has been successfully used to quantify several types of engineered nanoparticles (Koesslinger and Drost, 1998; Uttenthaler et al., 1998,2001; O'Sullivan and Guilbault, 1999; Klepeis et al., 2007). The QCM works via the piezoelectric effect and is capable of measuring and detecting changes in mass within the nanogram range (Mulhopt et al., 2009). QCMs have a working detection range for particle sizes between 150 and 500 nm (Desantes et al., 2006), which closely resembles the range for tobacco smoke particles (150-200 nm) making this technology ideal for the assessment of deposited tobacco particulate mass.

Recently and for the first time QCMs have been used to investigate the deposition of tobacco smoke particle mass in vitro. In fact, QCMs have been incorporated into several in vitro exposure chambers to assess real-time deposition uniformity/gradients and for quality control purposes (Adamson et al., 2012, 2013a,b). This is the first time that a dose tool has been able to measure deposited mass in real-time, in situ of exposure. Such a tool offers researchers the opportunities to investigate deposition data from a variety of cigarettes and potentially control exposure based on particulate

dose, simulating that of in vivo exposure. This in turn would make both the in vivo and in vitro data much more comparable. In a study presented by Adamson et al., in 2012 the authors accurately correlated deposition data from HPLC techniques and QCMs (R2 = 97.4%) demonstrating that these tools/methods can be used interchangeably. In a later study using QCMs, Adamson et al. (2013b) demonstrated the effect of airflow (l/min) on particle deposition in the Vitrocell® 6/4 CF Stainless module.

Irrespective of the exposure system, QCM tools offer researchers a consistent approach with an associated gravimetric unit of deposited mass per surface area (^g/cm2) that can be used to express in vitro data allowing more accurate comparisons between data from various systems to be made.

3.4. Vapour phase

Carbon monoxide, carbon dioxide and nitrogen oxides (CO, CO2 and NOx) are simple components of the complex vapour phase of tobacco smoke and due to their facile quantification by infrared gas analysers, make ideal smoke markers. Vapour phase markers can be quantified in-line or remotely, but both techniques have limitations. In-line quantification has the benefit of being in real-time; however it can only be applied to one smoke position/line (unless using more than one analyser). In addition, the analyser's flow rate may affect the smoke transit, altering results. Furthermore, actual concentrations appear as concentration peaks with every smoke puff, thus a mean exposure concentration over time must be calculated (Ritter et al., 2004). Alternatively, connecting smoke outlets to gas collection bags (Douglas bags) has the benefit that each line can be read after the smoke run. Furthermore, the gas being analysed is the total concentration amassed over the duration of the run, in a homogenous mixture. However, the duration of the smoke exposure run may need to be reduced significantly in order to prevent overfilling of the bag, in some cases high flow rates may not be possible at all.

More recently, Nara et al. (2013) demonstrated the ability to quantify carbonyls in tobacco smoke using a carbonyl DNPH (dini-trophenylhydrazine) trap and HPLC technique. Analysis of vapour phase components using chemistry trapping set-ups may provide the key in characterising vapour phase dilution and delivery within these systems. The vapour phase of smoke within these systems remains poorly understood considering it is the majority smoke fraction.

4. Discussion

For tobacco smoke generation, dilution and delivery in vitro, there are various commercial and bespoke whole smoke options available. These exposure technologies ensure that there are limited changes in the tobacco smoke material during collection, dilution, transit and delivery (Okuwa et al., 2010). Of the commercial systems available some have been characterised more than others in terms of published scientific literature and developed biological endpoints. Others are relatively new to the scientific field and are still establishing their presence. The Borgwaldt RM20S and associated exposure chamber have been almost exclusively used by British American Tobacco. The CULTEX® exposure modules have been used to assess a variety of airborne chemicals and are not limited to tobacco smoke. The Burghart Mimic Smoker MSB-01 offers a more high throughput based exposure, but cells are not exposed at the ALI which ultimately limits the use of this technology. The Vitrocell® VC 10 Smoking Robot has not been characterised to the extent of other systems, with very little information existing in the scientific domain, although this appears to be changing. Bespoke systems are also widely used for whole smoke exposure, but vary in smoke generation, dilution and exposure principles, that make comparison of these technologies difficult. What is clear is that they have a place in fundamental and mechanistic research. However, for a consistent approach these systems may be too unique in their individual development to be widely used. As yet, no exposure system commercially available or otherwise has been completely characterised or validated and each system offers unique advantages and disadvantages. Interestingly, an assessment of different whole smoke exposure technologies by the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) - a tobacco related in vitro task force, found remarkably consistent results, indicating these systems perform in a similar way (CORESTA air-liquid interface report). For example, reported losses in both the Burghart Mimic Smoker MSB-01 and Borgwaldt RM20S are similar at 40-50% (Scian et al., 2009a; Adamson et al., 2011). This is interesting considering these two set-ups are diverse from each other. What is clear, is that whole smoke exposure systems are an important development for the delivery of a physiologically relevant test smoke aerosol in vitro (Johnson et al., 2009). In support of this, in June 2009 the Committee on Mutagenicity in the UK reviewed the area of 'chemicals in foods, consumer products and the environment' and commented that the development of whole smoke exposure procedures were likely to provide more relevant data on the mutagenic activity of tobacco smoke, but noted none of the test systems had been "adequately validated" (Committee on Mutagenicity, 2009). Validation remains an obvious area for improvement in this field of research and as yet no one has conducted a multi-laboratory or multi-system study.

The scientific literature surrounding whole smoke in vitro systems has demonstrated a wealth ofassociated biological endpoints, disease and toxicological. For example, tobacco smoke in vitro has been shown to induce a variety of cellular effects potentially associated with disease processes, which include the up-regulation of a series of factors linked to lung damage and inflammation, tissue remodelling, mucin overproduction and cellular transformation (Leikauf et al., 2002; Breheny et al., 2005; Heeg et al., 2006; Olivera et al., 2007; Newland and Richter, 2008; Haswell et al., 2010). Tobacco smoke has also been shown to generate high levels of reactive oxygen species and oxidative stress which can cause cellular damage to lipids, proteins and DNA (Chow, 1993; Cooke et al., 2003; Federico et al., 2007). In addition, tobacco smoke has been shown to have multiple effects on gene expression in the human airways. Studies of bronchial epithelial cells obtained from the airways of smokers and non-smokers by bronchial brushing has indicated that cigarette smoke induces metabolising and redox

regulating genes, tumour suppressor genes and oncogenes alongside the regulation of inflammatory processes (Brody and Steiling, 2011; Hackett et al., 2011). Cellular glutathione response to tobacco smoke has also been extensively documented in in vitro and in vivo studies. The glutathione redox system is critical in maintaining intracellular glutathione levels, which are important to normal cellular physiological processes and antioxidant defence systems in the lung. If cellular glutathione levels are significantly altered a variety of cellular processes are initiated, such as the activation of the transcription factors AP-1 and Nuclear Factor kappa P, which can lead to the activation of a variety of disease pathways (Rahman and MacNee, 1999). In terms of in vitro test method development this is a promising sign, indicating that these systems have the potential to support and supplement a variety of future exposure scenarios.

5. Conclusions

The amount of smoke delivered to cells within in vitro exposure systems can be presented in many ways, often dependent upon the machine used, either as a ratio of smoke to air, or as a flow rate of mixing air applied to the smoke dilutor, or as a percentage or a smoke fraction. However, what is more relevant and is becoming increasingly important in the field of tobacco smoke assessment in vitro is dosimetry: i.e. how to quantify the amount of smoke cells are directly exposed to. At present there is no recognised approach to the measurement of dose, and the vapour phase of cigarette smoke within these systems remains poorly understood. With the variety of exposure options available to researchers and bespoke systems relatively easy to fabricate or replicate, dosimetry tools may bridge the gap and play an important role, not only in the measurement of actual cellular dose but also in the characterisation and validation of these systems. Furthermore, utilising dose tools such as the QCM in real-time will give confidence that the related exposure system is operating within expected limits, allow researchers to monitor exposure conditions, add strength to the resulting in vitro data, and allow cross-platform comparisons to be made where currently they cannot.

Advances in exposure technologies have allowed investigators to study some of the underlying mechanisms of tobacco induced cellular injury and ultimately disease mechanisms. Furthermore, a plethora of in vitro models have been developed alongside these exposure systems that may be used when assessing the biological activity of tobacco smoke. In terms of understanding tobacco smoke and disease, elucidating disease mechanisms and identifying smoke toxicants responsible for adverse health effects, in vitro methods will be key, providing the related exposure system is accurately characterised.

Declaration of interest

The authors report no declarations of interest and are employees of British American Tobacco.

Authors contributions

David Thorne conceived the article, reviewed the literature and drafted the manuscript. Jason Adamson contributed to the writing of the manuscript and produced all of the schematic illustrations and artwork contained within.

Acknowledgements

The authors would like to thank the following people at BAT: Derek Sharp for his technical assistance in the use of the ProQuest Dialogue platform, and Annette Dalrymple, Debbie Dillon, Clive

Meredith and Marianna Gaça for their time and efforts in reviewing this manuscript.

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A review of in vitro cigarette smoke exposure systems Academic research paper on "Environmental engineering"

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