Assessment of an in vitro whole cigarette smoke exposure system: The Borgwaldt RM20S 8-syringe smoking machine

Abstract

Background: There have been many recent developments of in vitro cigarette smoke systems closely replicating in vivo exposures. The Borgwaldt RM20S smoking machine (RM20S) enables the serial dilution and delivery of cigarette smoke to exposure chambers for in vitro analyses. In this study we have demonstrated reliability and robustness testing of the RM20S in delivering smoke to in vitro cultures using an in-house designed whole smoke exposure chamber.

Results: The syringe precision and accuracy of smoke dose generated by the RM20S was assessed using a methane gas standard and resulted in a repeatability error of ≤9%. Differential electrical mobility particle spectrometry (DMS) measured smoke particles generated from reference 3R4F cigarettes at points along the RM20S. 53% ± 5.9% of particles by mass reached the chamber, the remainder deposited in the syringe or connecting tubing and ~16% deposited in the chamber. Spectrofluorometric quantification of particle deposition within chambers indicated a positive correlation between smoke concentration and particle deposition. In vitro air-liquid interface (ALI) cultures (H292 lung epithelial cells), exposed to whole smoke (1:60 dilution (smoke:air, equivalent to ~5 μg/cm2)) demonstrated uniform smoke delivery within the chamber.

Conclusions: These results suggest this smoke exposure system is a reliable and repeatable method of generating and exposing ALI in vitro cultures to cigarette smoke. This system will enable the evaluation of future tobacco products and individual components of cigarette smoke and may be used as an alternative in vitro tool for evaluating other aerosols and gaseous mixtures such as air pollutants, inhaled pharmaceuticals and cosmetics.

Figure 1

Exposure chamber (left) patent publication number WO 03/100417 A1 and schematic cross-section (right), [7].

Figure 2

The Borgwaldt RM20S 8 syringe smoking machine. The dilutor syringes work independently, with the capability of delivering an extensive smoke dilution range (1:2 -1:4,000 [smoke:air, v/v]) and delivering up to eight different doses of whole smoke simultaneously. A - cigarette smoke generator; Bi - original 4-syringe unit; Bii - additional 4-syringe unit enabling 8 concurrent dilutions in a single run; C - a single BAT exposure chamber housed in an incubator at 37°C attached to smoke generator and media (up to 9 chambers can be installed in the incubator shown, one connected to each syringe plus an air control); D - cell culture media maintained at 37°C in an incubator, supplied to chambers using a pump; E - airflow fan controller.

Figure 3

The precision of eight individual syringes using hydrocarbon analysis. A methane gas standard was diluted by the eight Borgwaldt RM20S syringes and precision was calculated as a function of accuracy of dilution delivery. A - syringe precision at a dilution of 1:193 (smoke:air, v/v) against a target value of 520 ppm methane gas standard, n = 15. B - syringe precision at a dilution of 1:500 (smoke:air, v/v) against a target value of 200 ppm methane gas standard, n = 15, * = outliers.

Figure 4

Schematic cross-section of a single RM20S syringe and exposure chamber. Smoke was sampled at the exit of the syringe (α), pre-entry to chamber (β) and at the exit from the chamber (γ) using an electrical mobility spectrometer.

Figure 5

Assessment of cigarette smoke particle mass penetration through the RM20S system, using three different tubing types. An electrical mobility spectrometer sampled smoke at the exit of the syringe (α), before the chamber (β) and at the exit from the chamber (γ). Results expressed as percentage of average mainstream smoke (100% = 5.75 ± 0.31 mg) n = 3. Statistically significant differences were observed between the three sampling points (α, β, γ) for all tubing types tested, p = < 0.05, but no significant differences were observed between the three tubing types at each individual sampling point, p = 0.083.

Figure 6

Transwell particulate deposition (μg/cm²) at different smoke dilutions for a 30 minute whole smoke exposure.

Figure 7

Exposure chamber dosimetry investigated using NRU; cytotoxicity in H292 cells exposed to a smoke dilution of 1:60 (smoke:air, v/v) in 12 mm Transwells^® in a single exposure chamber. Dotted line represents 50% viability (EC₅₀). Mean viability = 46 ± 9.8%, p = 0.56, n = 20.