Assessment of cigarette smoke particle deposition within the Vitrocell® exposure module using quartz crystal microbalances

Abstract

Background: Cigarette smoking is a cause of a variety of serious diseases, and to understand the toxicological impact of tobacco smoke in vitro, whole smoke exposure systems can be used. One of the main challenges of the different whole smoke exposure systems that are commercially available is that they dilute and deliver smoke in different ways, limiting/restricting the cross-comparison of biological responses. This is where dosimetry - dose quantification - can play a key role in data comparison. Quartz crystal microbalance (QCM) technology has been put forward as one such tool to quantify smoke particle deposition in vitro, in real-time.

Results: Using four identical QCMs, installed into the Vitrocell® mammalian 6/4 CF Stainless exposure module, we were able to quantify deposited smoke particle deposition, generated and diluted by a Vitrocell® VC 10 Smoking Robot. At diluting airflows 0.5-4.0 L/min and vacuum flow rate 5 ml/min/well through the exposure module, mean particle deposition was in the range 8.65 ± 1.51 μg/cm(2)-0.72 ± 0.13 μg/cm(2). Additionally, the effect of varying vacuum flow rate on particle deposition was assessed from 5 ml/min/well - 100 ml/min/well. Mean deposited mass for all four airflows tested per vacuum decreased as vacuum rate was increased: mean deposition was 3.79, 2.75, 1.56 and 1.09 μg/cm(2) at vacuum rates of 5, 10, 50 and 100 ml/min/well respectively.

Conclusions: QCMs within the Vitrocell® exposure module have demonstrated applicability at defining particle dose ranges at various experimental conditions. This tool will prove useful for users of the Vitrocell® system for dose-response determination and QC purposes.

Keywords: Dosimetry; In vitro whole smoke exposure systems; Particle deposition; QCM; Quartz crystal microbalance; Tobacco smoke; Vitrocell®.

Figure 1

The Vitrocell VC 10 Smoking Robot and mammalian exposure module (6/4 CF Stainless) – a schematic cross-section. ([1]) Smoking robot carousel where cigarettes are loaded and smoked, enclosed in a ventilation hood. ([2]) Piston/syringe which draws and delivers ISO (35 ml) or Health Canada Intense (55 ml) mainstream cigarette smoke. ([3]) Air jets add continuous diluting air perpendicular to the mainstream smoke in the range 0.2-12 L/min; rates are set and maintained by mass flow controllers. ([4]) Dilution, transit and delivery of whole smoke occurs in the dilution bar, multiple parallel bars make up the dilution system. ([5]) Isolated cell culture inserts are supported and exposed to diluted whole smoke at the ALI in a module which docks under the dilution system; culture inserts can be removed and replaced with QCMs. ([6]) A vacuum (5–100 ml/min/well) is applied to the module which draws the diluted smoke from the dilution bar into the module via the ‘trumpet’ inlets. ([7]) Due to continuous diluting airflow, smoke remaining within the dilution system transits to exhaust away from the module.

Figure 2

The Vitrocell mammalian exposure module (6/4 CF Stainless). [A] Top view of the module base (lid removed) looking into the four separated wells where cell culture inserts would usually sit and be exposed to smoke, but where four identical QCM units have been installed, 1–4 (left-right). [B] A schematic cross-section of the module base and lid depicting how smoke is delivered to cell culture inserts/QCM. This illustration shows a very slight difference in the heights of the crystal surface and a typical cell exposed on a porous membrane; in our set-up the QCM is 1.5 mm higher than an exposed cell surface would be. However, the trumpet height can (and should) be adjusted to compensate for this when exposing cells and QCMs concurrently so that the gap distance is the same (2 mm in this case between crystal and trumpet).

Figure 3

Dose–response of QCM quantified whole smoke particle deposition in the Vitrocell 6/4 CF Stainless module. A multi-vari chart with individual values showing deposited particle mass for the 4 QCM positions (1–4, left-right) at diluting airflows ranging 0.5-4.0 L/min and sampling from the dilution system at 5 ml/min/well vacuum through the module (n = 5/QCM position/airflow).

Figure 4

QCM quantified whole smoke particle deposition in the Vitrocell module at various airflow (L/min) and vacuum (ml/min/well) rates. [A] A multi-vari chart showing deposition within the dose range of diluting airflows 0.5-4.0 L/min and at vacuum rates of 5–100 ml/min/well through the module (mean of 4 QCM positions per airflow). [B] A multi-vari chart showing the regional deposition across the module (separated by vacuum). The 4 connected black dots represent QCM positions 1–4, left to right (Figure 2A). Asterisks indicate statistically significant differences in deposition across/between module positions 1–4 as determined by one-way ANOVA: * denotes a statistically significant difference between positions, p = 0.05-0.01, ** denotes a statistically significant difference between positions, p = <0.01. For 5 ml/min/well vacuum, data sets were n = 5/QCM position/airflow; for 10, 50 and 100 ml/min/well vacuum, data sets were all n = 3/QCM position/airflow. For both charts, red diamonds indicate the mean deposited particle mass for all 4 airflows tested at each vacuum rate.