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Whole smoke

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Key messages

1. The whole smoke exposure system delivers biologically relevant doses of whole smoke to in vitro cultures supported at the air-liquid interface (ALI).
2. The exposure chamber can be used to assess the effects of smoke concentration, whole smoke vs. smoke gas phase, single aerosol or individual gas phase components.
3. The chamber can also be attached to other test generators, exposing in vitro models to gas and aerosol mixtures such as air pollutants and inhaled pharmaceuticals.
4. We wish to share our technology, making it available to other researchers to further the design and validation of physiologically relevant in vitro ALI culture systems.

Introduction

Historically, in vitro toxicological assessment of cigarette smoke has been based mainly on particulate matter which excludes the gas and vapours found in smoke. There have been limited advances over recent years to develop relevant in vitro exposure systems to study the toxicological affects of complex aerosols, including cigarette smoke. Bombick et al. have described a system in which cigarette smoke is directly introduced into a culture flask containing an adherent cell monolayer which is then placed on a rocking platform [1], where the in vitro cultures are exposed alternately to culture medium and smoke or gas phase. Studies indicate activity of whole smoke and gas phase in this system are comparable. 

The CULTEX™ system enables the exposure of in vitro cultures at the air-liquid interface (ALI) using a limited number of Transwell™ inserts.  Briefly, a maximum of three inserts are housed in a glass exposure device and cells at the ALI are exposed to a gaseous test compound apically whilst in contact with warmed culture medium basally. This system has been used to assess the cytotoxicity and genotoxicity of mainstream cigarette smoke [2,3]. Most recently Scian et al. have described the development and characterisation of an exposure system that uses a 96 well format, exposing in vitro cultures to 8 different dilutions of whole smoke replicated over six wells [4].

We have developed a novel whole smoke exposure system designed to expose in vitro cultures of human lung primary epithelial cells and cell lines cultured on Transwell™ inserts to mainstream whole cigarette smoke (UK patent publication WO 03/100417/ A1)[5]. This system allows all phases of cigarette smoke - particulate and gas - to be assessed separately or in combination. The chamber can be used to assess the effects of smoke concentration, whole smoke versus smoke gas phase as well as effects of single aerosol or individual gas phase components. Furthermore, this system could be a useful in vitro method for evaluating the effects of other aerosols and gaseous mixtures such as air pollutants and inhaled pharmaceuticals and to examine occupational exposure scenarios.

The exposure chamber

The chamber is a versatile and inexpensive device which can be produced in most engineering workshops. Made from clear durable autoclavable Perspex™ this allows the operator to view the contents of the chamber during exposure. It can be modified to accommodate a range of culture sizes and number (3 x 24mm inserts, or 6 x 12mm inserts, or 8 x 6mm inserts). The cells are cultured on Transwell™ inserts which are housed in the exposure chamber and are exposed apically to cigarette smoke. Fresh culture medium is introduced basally.  Contact between the media and the basal surface of the cells is regulated by dual peristaltic pumps which prevents flooding of the cell monolayer and disruption of the ALI. Media flow is unidirectional to avoid build up of smoke toxicants, which may affect exposure conditions basally. Cigarette smoke is exhausted into the chamber and old smoke is displaced by fresh smoke from the next puff.

The image below shows a schematic of a cross section of the whole smoke exposure chamber.

Our exposure chamber has the follwing design characteristics:-  

• Media flow is unidirectional and not recycled
• Cells are maintained basally without disrupting the air-liquid interface
• A distribution plate aids even delivery of the test aerosol
• Chambers are sealable with quick release mechanisms, which ensures an air-tight sterile environment suitable for cell culture purposes
• The chambers are Perspex and autoclavable, ensuring the chambers can be easily sterilised and cleaned

Whole smoke exposure system using Borgwaldt RM20s

Cigarette smoke is generated using a Borgwaldt RM20S smoke machine. The RM20S is a smoking machine that generates and dilutes cigarette smoke for in vitro cell culture investigations.

The machine was designed collaboratively with Borgwaldt-kc GmbH , a supplier of smoking engines to the tobacco industry, regulatory laboratories and independent testing laboratories. The RM20S is based on the rotary principle and having eight ports can smoke up to eight cigarettes simultaneously. The machine smokes the cigarettes as per ISO conditions, dilutes the smoke with filtered air via a syringe system 1:1 up to 1:5000 (smoke:air, v:v ratio) and delivers smoke to the exposure chamber to enable the direct exposure of ALI cell cultures. The RM20S is fully automatic and can smoke up to twenty cigarettes per port successively to supply the exposure chambers with diluted smoke over several hours. Using incubators, the chamber is maintained at 37°C and fed with a continuous supply of pre-warmed fresh culture medium throughout the exposure period (30 minutes to 3 hours).  This ensures that endogenous stress levels within the cells are not artificially elevated. Stale smoke is expelled from the chamber by active displacement, upon delivery of freshly generated smoke.

The image below shows the whole smoke exposure system.

Dosimetry

A number of in-house studies have been conducted to investigate and characterise cigarette smoke within our smoke exposure system. Research using an electrical mobility spectrometer has investigated smoke particle losses and changes in particle size distribution through the system. Measurements were taken at three points in the system: after smoke generation from the Borgwaldt RM20S, at delivery to the chamber and at the exhaust from the chamber. These studies indicate 80-95% of particulates reach the chamber with 33% being deposited in the chamber. Assessment of particle deposition on inserts and cell cytotoxicity within the chamber indicates smoke particle diameter ranging from 100-300 nm, delivery within chambers to be at biologically relevant doses (0.1-5 µg particulates/cm2 per cigarette) and uniform whole smoke delivery within the chamber across inserts.

Post exposure applications

This exposure system enables us to investigate effects of whole cigarette smoke on endpoints considered to be relevant to major smoking related disease, namely cancer and chronic obstructive lung disease (chronic bronchitis and emphysema). Endpoints we have measured in NCI-H292 human lung epithelial cells include the effects of serial dilutions of smoke on cell viability [5], oxidative DNA damage (as measured using the Comet assay) [6], gene expression and cytokine production of putative disease-related mediators [5,7] For more information on the comet assay please see the Cancer and COPD pages. 

Results from experiments using this whole smoke exposure system demonstrate a dose dependent decrease in cell viability with a decrease in smoke dilution.  Products designed to give different deliveries of particulate and gas phase constituents gave significantly different dose response curves.  Exposure of cell cultures to smoke gas phase only, indicated that in our exposure system ~ 80% of the cytotoxic effect was from the gas phase constituents and ~20% from the particulate phase [8]. 

The schematic above shows: A - The first puff of the cigarette is taken to ISO standards, with sterile air drawn in to dilute the ISO puff; B - The syringe exhausts the excess cigarette smoke into a separate line away from the exposure chamber; C - Sterile air is again drawn into the syringe to obtain the desired dilution; D - the desired dilution of cigarette smoke is exhausted into the chamber. An in-line Cambridge filter pad can trap the majority of particulates and some semi-volatile components from the puff. In the Schematic above, chambers A and B are delivered diluted mainstream cigarette smoke, chambers C and D are delivered diluted primarily particulate-free mainstream cigarette smoke. This system allows the assessment of the various phases of mainstream smoke together of independently.

We have worked with others through a CORESTA task force to investigate the in vitro response to smoke of cells at the ALI [9].

Future studies

We are currently developing the capability to generate smoke using the Vitrocell VC10 smoking robot , a versatile novel smoking engine. The Vitrocell  VC10 smoking robot has been designed purely for basic research and is very adaptable; it offers the generation of whole smoke to in vitro cultures using a broader range of smoking regimes, puff by puff analysis, in line particulate analysis and importantly is compatible with our smoke exposure chamber.

1. Bombick, D.W., Ayres, P.H., Doolittle, D.J. (1997). Cytotoxicity assessment of whole smoke and vapour phase of mainstream and sidestream cigarette smoke from three Kentucky reference cigarettes. Toxicological Methods. 7: 177-190.
2. Ritter, D., Knebel, J.W., Aufderheide, M. (2001). In vitro exposure of isolated cells to native gaseous compounds- development and validation of an optimised system for human lung cells. Experimental Toxicology and Pathology. 53:373-386.
3. Aufderheide, M., Knebel, J.W., Ritter, D. (2003). An improved in vitro model for testing the pulmonary toxicity of complex mixtures such as cigarette smoke. Experimental Toxicology and Pathology. 55:51-57.
4. Scian, M.J., Oldham, M.J., Kane, D.B., Edmiston, J.S., McKinney, W.J. (2009). Characterisation of a whole smoke in vitro exposure system (Burghart Mimic Smoker-01). Inhalation Toxicology. 21:3, 234-243.
5. Phillips, J., Kluss, B., Richter, A., Massey, E. D. (2005). Exposure of bronchial epithelial cells to whole cigarette smoke: assessment of cellular responses. ATLA. 33 (3): 239-248. Link to abstract and citation. 
6. Thorne, D., Wilson, J., Kumaravel, T.S., Massey, E.D., McEwan, M. (2009). Measurement of oxidative DNA damage induced by mainstream cigarette smoke in cultured NCI-H292 cells. Mutation Research. 673 (1): 3-8. Link to abstract and citation. 
7. Maunders, H., Patwardhan, S., Phillips, J., Clack, A., Richter, A. (2007). Human bronchial epithelial cell transcriptome: Gene expression changes following acute exposure to whole cigarette smoke in vitro. American Journal of Physiology - Lung Cellular and Molecular Physiology. 292 (5): L1248-L1256. Link to poster. 
8. Phillips, J., Richter, A., Massey, E. D. (2004). Determination of the cytotoxicity of whole cigarette smoke. Poster presented at the Annual Congress of the British Toxicological Society, Heriot Watt University, Edinburgh, April 21-24, 2004. Link to poster. 
9. CORESTA In Vitro Toxicology Task Force. In vitro exposure of cells to smoke at the air liquid interface .