Collecting e‐cigarette aerosols for in vitro applications: A survey of the biomedical literature and opportunities to increase the value of submerged cell culture‐based assessments

Abstract

Electronic nicotine delivery systems (ENDS) are being developed as potentially reduced-risk alternatives to the continued use of combustible tobacco products. Because of the widespread uptake of ENDS-in particular, e-cigarettes-the biological effects, including the toxic potential, of their aerosols are under investigation. Preclinically, collection of such aerosols is a prerequisite for testing in submerged cell culture-based in vitro assays; however, despite the growth in this research area, there is no apparent standardized collection method for this application. To this end, through an Institute for in vitro Sciences, Inc. workshop initiative, we surveyed the biomedical literature catalogued in PubMed® to map the types of methods hitherto used and reported publicly. From the 47 relevant publications retrieved, we identified seven distinct collection methods. Bubble-through (with aqueous solvents) and Cambridge filter pad (CFP) (with polar solvents) collection were the most frequently cited methods (57% and 18%, respectively), while the five others (CFP + bubble-through; condensation; cotton filters; settle-upon; settle-upon + dry) were cited less often (2-10%). Critically, the collected aerosol fractions were generally found to be only minimally characterized chemically, if at all. Furthermore, there was large heterogeneity among other experimental parameters (e.g., vaping regimen). Consequently, we recommend that more comprehensive research be conducted to identify the method(s) that produce the fraction(s) most representative of the native aerosol. We also endorse standardization of the aerosol generation process. These should be regarded as opportunities for increasing the value of in vitro assessments in relation to predicting effects on human health. K E Y W O R D S aerosol collection, e-cigarette, in vitro assays, literature survey, submerged cell cultures 1 | INTRODUCTION Electronic nicotine delivery systems (ENDS) are being developed as potentially reduced-risk alternatives to the continued use of combustible tobacco products (Brandon et al., 2015; Farsalinos & Polosa, 2014). Electronic cigarettes (e-cigarettes)-one of the most widely known ENDS-consist of a battery-powered device that heats an "e-liquid" contained inside an atomizer, which leads to generation

The in vitro ToxTracker and Aneugen Clastogen Evaluation extension assay as a tool in the assessment of relative genotoxic potential of e-liquids and their aerosols

“…The bubbling of smoke/aerosol through an aqueous solution (e.g. PBS, cell culture medium) to achieve in vitro exposure has been used in various studies ( 27 , 30 , 46–48 ). This trapping method accounts for the water-soluble fraction of the smoke/aerosol, which is thought to be representative of the fraction soluble in the blood ( 47 ) and also allows a means of exposure to cells that must be submerged in medium.…”

In vitro determinants of e-cig aerosol condensate bioavailability and toxicity: Influence of cell culture parameters and utility of a normalized dose metric in e-cigarette studies

“…Attempts to synthesize the information available from the larger body of e-cig toxicological data are already fraught with complications. The complicating factors include: the sheer number of commercially available e-liquid formulations (over 8,000 unique flavors and counting [63]), differences in e-cig device design and functionality [59, 64], the highly variable vaping protocols used for e-cig aerosol generation [12], and the choice of in vitro cell culture platforms available for respiratory research, i.e. two-dimensional submerged cultures or three-dimensional air-liquid interface cultures [65].…”

“…two-dimensional submerged cultures or three-dimensional air-liquid interface cultures [65]. Calls have been made for the development of standardized research protocols to subvert these issues [12, 66–69]. We propose that such protocols should additionally include standardized dose reporting methods.…”

“…Preclinical studies of e-cigs rely heavily, for obvious reasons, on in vitro cell culture systems. There is, however, a pervasive lack of consistency in the research methodologies used, ranging from the type of e-cig device used to the methods employed for e-cig aerosol generation and subsequent in vitro delivery to cells [12]. These differences can confound the comparisons of results from different studies or laboratories, thereby limiting the combined utility of the generated data.…”

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Predict the Cardiotoxicity Potential of Next Generation Nicotine Products

“…To allow for the in vitro exposure of cigarette smoke and NGP aerosols, a number of trapping methodologies have been employed ( Smart and Phillips, 2021 ). The bubbling of whole smoke or aerosol in phosphate buffered saline (bPBS) is a commonly used method and allows for the direct addition of bPBS into an in vitro cell system ( Johnson et al, 2009 ).…”