Toxicol In Vitro. 2017 Feb;38:150-158.

A new fluorescence-based method for characterizing in vitro aerosol exposure systems


Sandro Steiner PhD, Shoaib Majeed MSc, Gilles Kratzer MSc, Julia Hoeng PhD, Stefan Frentzel PhD

Philip Morris International R&D, Philip Morris Products S.A. (part of Philip Morris International group of companies)

Quai Jeanrenaud 5, CH-2000 Neuchatel, Switzerland


Corresponding author

Sandro Steiner

Philip Morris International R&D

Quai Jeanrenaud 5

CH-2000 Neuchâtel


Tel: +41 (0)58 242 23 84


PubMed link



There is an increasing interest in the pharmacological testing of inhalable therapeutics and the toxicological assessment of aerosol generating consumer products such as electronic cigarettes. In light of the attempts to reduce animal experimentation, this is projected to involve more and more in vitro methodologies. In vitro assessment of aerosols is technically challenging, as for realistically simulating the interaction between a native aerosol and the epithelial lining of the respiratory tract, aerosol exposures need to be conducted at the air-liquid interface (ALI), that is, cell cultures need to be brought into direct contact with the test aerosol. In order to do this under controlled conditions, aerosol exposure systems have been developed, which allow i) keeping cell cultures in a defined environment, ii) conditioning of the test aerosol, for instance with respect to temperature, relative humidity and concentration and iii) delivering the aerosol to the cell cultures [1, 2].

State of the art exposure systems share the common limitation that they may change, for instance, the particle number-size distribution of a test aerosol during its passage through the system [3]. In addition, the efficiency by which (semi-)volatile and particulate aerosol components or particles of different sizes are delivered to the cell cultures may not be equal [4-7]. As a consequence, knowledge of the dose of the test aerosol supplied to the exposure system is not sufficient for describing the exposure, unless it is known how the system translates this applied dose into a delivered dose, i.e. what fraction of the applied aerosol ultimately reaches and interacts with the cell cultures.

Optimally, the dose delivery is monitored simultaneously with cell culture exposures, for instance by exposing samples of a trapping solvent along with the cell cultures, followed by quantification of aerosol constituents in the samples. For setting up new exposure conditions and for exposure system characterization, independent test exposures may be preferred however, as they allow using labeled model aerosols that would not be feasible for cell exposures, but can, upon deposition in the system, be retrieved and quantified reliably, fast and cost-efficiently.



We developed a method for the quantification of aerosol deposition that, because of its simple but robust character, is applicable even if extensive analytical equipment is unavailable. We used glycerol as aerosol material, since it allows generating stable aerosols and is one of the main constituents of the liquids vaporized in electronic cigarettes and several medical devices for drug inhalation [8-11].



Aerosols of different particle size distributions and low geometric standard deviations were generated in a TSI Condensation Monodisperse Aerosol Generator (CMAG), by condensing glycerol on Aitken mode particles consisting of the fluorophore disodium fluorescein (DSF). The stability of aerosol size distributions over time was assessed using a TSI Aerodynamic Particle Sizer, the stability of aerosol mass flow rates by trapping the aerosols on Cambridge filters followed by gravimetric determination of the collected mass. The DSF content of the aerosols was determined by elution of the material collected on the filters in phosphate buffered saline (PBS) and the fluorometric quantification of DSF in the obtained eluates.

The aerosols were then used for test exposures in the Vitrocell® 24/48 aerosol exposure system. For this purpose, 100 µL of PBS were pipetted into 24-well sized cell culture inserts and exposed in the system during defined periods of time. Upon exposures, the PBS samples were retrieved from the cell culture inserts and their DSF contents were determined fluorometrically.

By putting the DSF content of the exposed PBS samples into relation with the DSF content of the aerosols used for exposures, aerosol mass deliveries as well as aerosol delivery efficiencies could be derived.


Results and Discussion

In course of this work, four aerosols of different particle size distributions (0.8, 1.1, 1.4 and 1.6 µm mean aerodynamic diameter, geometric standard deviations of 1.3 – 1.4) were generated and used for test exposures. The characterization of the aerosols revealed that they could be generated reproducibly with a high stability of relevant aerosol parameters, i.e. particle size distribution, aerosol mass flow and aerosol DSF content.

Quantification of DSF in exposed PBS samples demonstrated the robustness, reliability and in particular the high sensitivity of the method. The lower limit of quantification for DSF in PBS was at 0.1 µg/L, which, even for the aerosol of the lowest DSF content, allowed detecting as little as 15 µg aerosol per exposed cell culture insert. DSF is highly water soluble, a retention of the fluorophore in cell culture inserts and a consequential bias in its quantification could be ruled out. In addition, it could be quantitatively removed from internal surfaces of the aerosol exposure system, which not only minimizes the risk of carry-over between individual exposures, but also opens the possibility of quantifying aerosol losses within the system.



We describe a method for aerosol exposure system characterization that relies on fluorescent liquid model aerosols of tunable mean particle sizes. We demonstrate that the model aerosols are stable, can be generated reproducibly and upon deposition in an aerosol exposure system, can be quantitatively retrieved from the system and quantified in a time and cost efficient way with high sensitivity and robustness. Although the method was specifically developed for exposure system characterization, its robustness and its ease of application render it a valuable tool for quantification of aerosol deposition in a broader context, e.g. for the experimental confirmation of computationally simulated particle dynamics or the description of aerosol delivery in test systems of high complexity such as models of the human respiratory tract.



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