Title: 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: Journal of Applied ToxicologyVolume 41, Issue 1 p. 161-174 REVIEW ARTICLEOpen Access 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 Daniel J. Smart, Corresponding Author Daniel J. Smart [email protected] orcid.org/0000-0002-7531-3584 PMI R&D, Philip Morris Products SA, Neuchâtel, Switzerland Correspondence Dr. Daniel J Smart, PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchâtel, Switzerland. Email: [email protected] for more papers by this authorGary Phillips, Gary Phillips Imperial Brands PLC, Bristol, UKSearch for more papers by this author Daniel J. Smart, Corresponding Author Daniel J. Smart [email protected] orcid.org/0000-0002-7531-3584 PMI R&D, Philip Morris Products SA, Neuchâtel, Switzerland Correspondence Dr. Daniel J Smart, PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchâtel, Switzerland. Email: [email protected] for more papers by this authorGary Phillips, Gary Phillips Imperial Brands PLC, Bristol, UKSearch for more papers by this author First published: 04 October 2020 https://doi.org/10.1002/jat.4064Citations: 2AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat 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. 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 of an inhalable aerosol upon puffing by the user (McRobbie, Bullen, Hartmann-Boyce, & Hajek, 2014). E-cigarette devices can vary extensively in terms of design and functionality (Breland et al., 2017). Similarly, e-liquids are also highly diverse and can contain different levels of nicotine, flavoring agents, and humectants such as propylene glycol and vegetable glycerin (Brown & Cheng, 2014). The recent rise in the use of e-cigarettes around the world has brought the toxicity of their aerosols into focus (Callahan-Lyon, 2014; Orr, 2014). Many institutes, including those from industry, academia, and government, are conducting research to understand their toxicological hazard and risk potential. As in other areas, much of the preclinical research on e-cigarette-derived aerosols is performed in in vitro cell culture models because they are relatively inexpensive (compared with animals), amenable to different types of higher-throughput analyses, supportive of the 3Rs principles (to Replace, Reduce, and Refine animal usage in scientific experiments), and importantly, the data generated from these models are potentially translatable to higher levels of biological organization. Significantly, in vitro toxicology data can influence the development of an e-cigarette device or e-liquid formulation. However, because many in vitro assays are conducted in submerged two-dimensional cell cultures, the aerosol generated from an e-cigarette must first be collected before it can be applied to the cell model under investigation. This challenge was originally faced by researchers seeking to investigate the toxicity of cigarette-derived smoke in vitro (Bradford, Harlan, & Hanmer, 1936). Ultimately, relatively standardized processes were developed whereby the smoke from combustible tobacco products was generated via smoking machines and subsequently collected in several ways (reviewed in Klus, Boenke-Nimphius, & Müller, 2016), including (a) total particulate matter or condensate captured on a Cambridge (glass fiber) filter pad (CFP) and desorbed with dimethyl sulfoxide (DMSO); (b) condensate captured via electrostatic precipitation (EP) and solubilized in DMSO; (c) condensate captured in a cold trap; and (d) aqueous solution (AQ)-soluble gas–vapor phase (GVP) constituents captured in phosphate-buffered saline (PBS). Some of these collection methods can be applied in tandem—for example, sequential CFP- or EP- and AQ-mediated trapping—and they can produce fractions that are broadly representative of the composition of tobacco smoke when considered as a whole (Klus, Boenke-Nimphius, & Müller, 2016). Although parallels can be drawn between cigarettes and e-cigarettes in the context of smoke and aerosol collection for in vitro applications, the latter products are more contemporary than the former and, consequently, have not been subjected to the same degree of experimentation. Thus, the general level of knowledge that has been built over the decades in relation to smoke generation and collection, at present, exists only minimally for e-cigarette-derived aerosols. Hence, there might be scope to ameliorate various aspects of the procedures linked to e-cigarettes. The Institute for in vitro Sciences, Inc. (IIVS) is currently hosting a series of workshops that provide a forum for stakeholders to identify, discuss, and develop recommendations for optimal generation of test samples and use of genetic toxicology in vitro assays to support tobacco product regulatory requirements (Moore et al., 2020). This workshop series follows two previous IIVS workshops that focused on in vitro models for chronic obstructive pulmonary disease and in vitro exposure systems and related dosimetry (Behrsing et al., 2016; Behrsing et al., 2017). Because evaluation of ENDS represents a new challenge for in vitro testing, practical issues associated with these products are a major focus of the current IIVS workshop series. During the initial workshop, the participants agreed on the need for reviewing the state of the science in relation to e-cigarette aerosol collection for in vitro applications, and, predicated on this consensus view, a survey of the biomedical literature was conducted in order to map the types of methods employed for this purpose. The present publication provides a summary of this survey. It should be noted that in vitro systems composed of cells cultured at the air–liquid interface coupled with whole aerosol exposure technologies were out of scope for this survey because it is a highly specialized area of research and merits its own dedicated review. Importantly, the opportunities arising from this survey should be exploited to improve the understanding and study of e-cigarette-derived aerosols in submerged cell culture-based in vitro assays. 2 METHODOLOGY 2.1 Literature search We conducted a search via PubMed® (https://www.ncbi.nlm.nih.gov/pubmed/)—the freely accessible literature repository containing >30 million publications from the fields of biomedicine and health (PubMed, 2020)—to identify all potentially relevant publications for subsequent evaluation. The most recent search was conducted during December 2019. We used the following search terms: (("electronic cigarette"[All Fields] OR "electronic cigarettes"[All Fields]) OR "e-cigarette"[All Fields]) OR ((("electronic nicotine delivery systems"[MeSH Terms] OR ((("electronic"[All Fields] AND "nicotine"[All Fields]) AND "delivery"[All Fields]) AND "systems"[All Fields])) OR "electronic nicotine delivery systems"[All Fields]) OR "e cigarettes"[All Fields]). Results were filtered by year (2013–2019: the time period when the vast majority of these publications was published) and reviews were excluded. Publications were further triaged by evaluating their abstracts (exported from PubMed®) for the following keywords: Aerosol; Capture; Collection; Condensate; Emissions; Immobiliz(s)ation; In vitro; Oxidative; Toxicity; Toxicology; Trapping; Vapo(u)r. 2.2 Data extraction, compilation, and visualization Triaged publications were critically evaluated for the presence of empirical information related to collection of e-cigarette aerosols for evaluation in in vitro assays. Relevant data were subsequently extracted and used to compile a database; the categories of data extracted are described in Tables 1 and 2. Note that, in publications that assessed more than one "item," the items were grouped together for entry into the database. For instance, a publication that evaluated 39 e-liquids and 5 e-cigarette devices via two different vaping regimens would be represented in the relevant fields of the database as "39 Types," "5 Types," and "2 Types," respectively. In addition, database fields were completed as "not available" (N/A), where relevant information was lacking or not explicit. Data were visualized by using Spotfire® Desktop (v7.13.0, TIBCO®, Palo Alto, CA, USA). TABLE 1. Selected database statistics Parameter Details Publications 47 Individual collection methods 49 Primary institutes ≥34 Publication year range 2013–2019 Research areas Immunology; inflammation; oral health; oxidative stress; tissue repair; toxicology; vascular TABLE 2. Summary of the collection method-related information from the 47 publications and additional data Collection method Manuscript reference PMID Collection solvent(s) Fraction(s) E-cigarette device E-liquid Smoking machine Puffs Vaping regimen Nicotine quantified Bubble-through Breheny, Oke, Pant, & Gaça, 2017 28 444 993 Medium AQE Vype ePen Blended Tobacco SM-450 10 CRM No. 81 Yes Rayner, Makena, Prasad, & Cormet-Boyaka, 2019 31 166 129 Medium AQE Innokin VV4/Nautilus tank Tobacco row N/A N/A 55-mL vol, 5-s draw, 30-s interval Yes Munakata et al., 2018 30 227 175 Medium AQE 2 types N/A N/A 300 HCI Yes Taylor et al., 2017 28 658 606 Medium AQE 2 types Blended tobacco RM20H 10 CRM No. 81 Yes Bengalli, Ferri, Labra, & Mantecca, 2017 29 053 606 Medium AQE Kit iSimple Ribilio/C14 Passthrough 12 types TRUST-iCERT 200 55-mL vol, 3-s draw, 60-s interval No Behar et al., 2016 27 633 763 Medium AQE 2 types 39 types N/A 24 2 types No Farsalinos et al., 2013 24 135 821 Medium AQE 2 types 21 types Vacuum N/A 2 types No Ganapathy et al., 2017 28 542 301 HEPES-buffered saline AQE 2 types 5 types Vacuum N/A HCI Yes Rubenstein, Hom, Ghebrehiwet, & Yin, 2015 26 072 673 HEPES-buffered saline AQE 2 types 3 types Vacuum N/A N/A No Ji et al., 2016 28 033 425 Medium AQE N/A 4 types Homemade N/A 33- to 83-mL vol, 2- to 5-s duration No Anderson, Majeste, Hanus, & Wang, 2016 27 613 717 Medium AQE 4 types Tobacco Vacuum N/A 55-mL vol, 2-s duration, 30-s interval No Teasdale, Newby, Timpson, Munafò, & White, 2016 27 137 404 Medium AQE Aerotank Mini/iStick battery Haven fluid USA Mix N/A 5 5.8-mL vol, 5-s draw, 10-s interval Yes Taylor et al., 2016 27 690 198 Medium AQE 2 types Blended tobacco RM20H 10 CRM No. 81 Yes Omaiye, McWhirter, Luo, Pankow, & Talbot, 2019 30 896 936 Medium AQE JUUL EC 8 types Peristaltic pump N/A 43- to 56-mL vol, 4.3-s draw, 60-s interval Yes Leslie et al., 2017 28 470 141 Medium AQE 5 types 15 types Diaphragm pump 14 ISO No Rankin et al., 2019 30 957 912 Medium AQE Joyetech eVic VT/eGo ONE Mega atomizer 2 types Water aspirator 13 1.5-s draw, 30-s interval No Kaisar, Sivandzade, Bhalerao, & Cucullo, 2018 29 879 439 PBS AQE N/A N/A SCSM 8 FTC No Bharadwaj, Mitchell, Qureshi, & Niazi, 2017 27 875 752 Medium AQE N/A Classic tobacco Vacuum N/A N/A Yes Yu et al., 2015 26 547 127 Medium AQE 2 types 4 types N/A N/A N/A No Di Biase, Attorri, Di Benedetto, & Sanchez, 2018 30 575 566 FBS AQE Kelvin 2 types Vacuum N/A 10-s draw No Higham et al., 2016 27 184 092 Medium AQE 3 types 6 types Peristaltic pump N/A 2 types No Higham, Bostock, Booth, Dungwa, & Singh, 2018 29 615 835 Medium AQE V5/CE5 clearomiser/VIP battery USA tobacco Peristaltic pump N/A N/A No Hom et al., 2016 27 096 416 HEPES-buffered saline AQE 2 types 5 types Vacuum N/A N/A No Otręba, Kośmider, Knysak, Warncke, & Sobczak, 2018 29 665 082 Medium AQE eGo-3 twist battery/bottom headed clearomizer 6 types Palaczbot 30 70-mL vol, 1.8-s draw, 17-s interval No Raez-Villanueva, Ma, Kleiboer, & Holloway, 2018 30 048 688 Medium AQE EVOD Kanger-Tech 2 types N/A N/A N/A Yes Ween, Whittall, Hamon, Reynolds, & Hodge, 2017 28 867 672 Medium AQE EVOD-2 10 types N/A 50 3-s draw, 5-s interval No Zahedi et al., 2019 31 200 115 Medium AQE N/A 2 types UoK ASM N/A 4.3-s draw, 60-s interval No Zhao et al., 2018 29 102 637 Medium AQE N/A 2 types ECAG N/A 2 types Yes CFP Breheny, Oke, Pant, & Gaça, 2017 28 444 993 DMSO DMSO-sol ACM Vype ePen Blended tobacco LM20X 40 CRM No. 81 Yes Rayner, Makena, Prasad, & Cormet-Boyaka, 2019 31 166 129 Medium AQ-sol ACM Innokin VV4/Nautilus tank Tobacco row N/A N/A 55-mL vol, 5-s draw, 30-s interval Yes Thorne et al., 2016 27 908 385 DMSO DMSO-sol ACM Vype ePen Blended tobacco LM20X N/A CRM No. 81 No Misra, Leverette, Cooper, Bennett, & Brown, 2014 25 361 047 PBS AQ-sol ACM blu eCigs 4 types Vitrocell VC10 N/A HCI Yes Husari et al., 2016 26 272 212 Medium AQ-sol ACM V4L CoolCart/Vapor Titan Soft Touch battery Strawberry ONARES N/A 80-mL vol, 4-s duration, 14-s interval No Shaito et al., 2017 29 079 789 Medium AQ-sol ACM V4L CoolCart Strawberry ONARES N/A 80-mL vol, 4-s duration, 14-s interval No Dalrymple et al., 2018 30 346 667 DMSO DMSO-sol ACM NVP Twilight tobacco LM20X 200 CRM No. 81 No Ito et al., 2019 31 400 404 DMSO DMSO-sol ACM Vype ePen Blended tobacco RM20D N/A CRM No. 81 Yes Thorne et al., 2019 31 163 219 DMSO DMSO-sol ACM N/A N/A RM200a 60 CRM No. 81 Yes CFP + bubble-through Takahashi et al., 2018 29 158 044 DMSO + PBS DMSO-sol ACM + AQ-sol GVP NTV N/A Rotary 70 HCI No Condensation Scott et al., 2018 30 104 262 N/A Conden-sate Kanger 2 types N/A N/A 3-s draw, 30-s interval Yes Clapp et al., 2017 28 495 856 N/A Conden-sate LAVABOX DNA 200/SMOK TFV4/TF-CLP2 Clapton coil 7 types Peristaltic pump 20 30-s Interval No Lei, Lerner, Sundar, & Rahman, 2017 28 256 533 N/A Conden-sate eGO/eGo Vision Spinner battery 4 types Peristaltic pump N/A 4-s draw, 30-s interval No Schweitzer et al., 2015 25 979 079 N/A Conden-sate Innokin iClear 16 3 types Vacuum N/A N/A No Sun, Kosinska, & Guttenplan, 2019 31 373 329 25% DMSO/Water/PBS AQ-sol condensate 2 types 2 types ASPECG pump N/A ISO Yes Cotton filters Miyashita et al., 2018 29 437 942 PBS AQ-sol ACM RBC CE5 Clearo-mizer 2 types Peristaltic pump 25 N/A No Settle-upon Shivalingappa, Hole, Westphal, & Vij, 2016 26 377 848 Medium AQE Kanger EVOD Flavorless Motor N/A N/A No Behar, Wang, & Talbot, 2018 28 596 276 Medium AQE iClear16D dual coil/Innokin iTaste MVP 3.0 battery 45 types Peristaltic pump N/A 56-mL vol, 4.3-s draw, 60-s interval No Alanazi, Park, Chakir, Semlali, & Rouabhia, 2018 29 800 583 Medium AQE EMOW Smooth Canadian tobacco Peristaltic pump N/A 10-s draw, 30-s interval No Zahedi, Phandthong, Chaili, Remark, & Talbot, 2018 30 032 837 Medium AQE N/A 2 types N/A N/A 4.3-s draw, 60-s interval No Settle-upon + dry Tommasi, Bates, Behar, Talbot, & Besaratinia, 2017 29 191 599 MtOH/DMSO MtOH-/DMSO-sol extract 3 types N/A Peristaltic pump 10 3 types No Abbreviations: ACM, aerosol collected matter; ASPECG, aerosol single port electronic cigarette generator; AQ, aqueous solution; AQE, aqueous extract; CRM No. 81, CORESTA recommended method number 81 (square wave puff profile, 55-mL vol, 2-s draw, 28-s interval); DMSO, dimethylsulfoxide; ECAG, e-cigarette aerosol generator; FTC, Federal Trade Commission (bell-shaped puff profile, 35-mL vol, 2-s draw, 58-s interval); FBS, fetal bovine serum; GVP, gas–vapor phase; HCI, Health Canada Intensive (bell-shaped puff profile, 55-mL vol, 2-s draw, 28-s interval); HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; ISO, International Organization for Standardization 3308, (bell-shaped puff profile, 35-mL vol, 2-s draw, 58-s interval); MtOH, methanol; N/A, not available; NTV, novel tobacco vapor product; NVP, novel vapor product; ONARES, oro-nasal respiratory exposure system; PBS, phosphate-buffered saline; SCSM, single cigarette smoking machine; s, seconds; sol, soluble; vol, volume; UoK ASM, University of Kentucky analytical smoking machine. 3 RESULTS 3.1 General database statistics The initial search retrieved 4561 publications. From these, further keyword triaging identified 1543 publications. Upon inspection, 47 publications from the 1543 were found to contain relevant empirical data, while the remaining were rejected because of lack of direct relevance to e-cigarette aerosol collection for in vitro application and/or absence of empirical information. Interestingly, two of these publications reported two distinct collection methods each (Breheny et al., 2017; Rayner et al., 2019). Thus, in total, there were 49 individual collection methods itemized in the database. Selected database statistics are described in Table 1. 3.2 Collection method-related information Table 2 provides a summary of the relevant data. Seven distinct collection methods were reported in the 47 publications; these were defined as "bubble-through," "CFP," "CFP + bubble-through," "condensation," "cotton filters," "settle-upon," and "settle-upon + dry." Each collection method is described in the Section 4, while graphical illustrations are presented in another publication emanating from the IIVS workshop series (Wieczorek et al., 2020). Bubble-through and CFP were the most frequently cited collection methods (57% and 18%, respectively), while the others were cited less often (2–10%) (Figure 1). FIGURE 1Open in figure viewerPowerPoint Types of methods employed for collection of e-cigarette-derived aerosols for in vitro research. CFP, Cambridge filter pad In addition, eight different solvent systems were used in these collection methods, including "25% DMSO/Water/PBS," "DMSO," "DMSO + PBS," "fetal bovine serum (FBS)," (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES)-buffered saline," "medium," "methanol (MtOH)/DMSO," and "PBS" (Table 2). Note that four studies applied the "condensation" collection method without a solvent and, thus, were represented with N/A in these fields of the database. Consequently, on the basis of the collection methods and solvents employed in the 47 publications, we defined seven different categories of aerosol fraction(s): "AQ-soluble aerosol collected matter (ACM)," "AQ-soluble condensate," "aqueous extract (AQE)," "condensate," "DMSO-soluble ACM," "DMSO-soluble ACM + AQ-soluble GVP," and "MtOH-/DMSO-soluble extract" (Table 2). A graphical summary of this information is provided in Figure 2. FIGURE 2Open in figure viewerPowerPoint Solvents used in collection of e-cigarette aerosols and, consequently, the fraction(s) evaluated in in vitro research. AQ, aqueous solution; AQE, aqueous extract; ACM, aerosol collected matter; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; GVP, gas–vapor phase; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MtOH, methanol; N/A, not available; PBS, phosphate-buffered saline; sol, soluble 3.3 Additional findings In general, there was large heterogeneity among the other e-cigarette aerosol generation-related parameters in the 47 publications. For example, the collected aerosols were generated from a multitude of e-cigarette devices and e-liquids via numerous different commercially available smoking machines or laboratory-built apparatuses (Figure 3). Furthermore, there was also diversity in the vaping regimens and number of puffs applied for aerosol generation (Figure 4). In addition, a number of studies performed very limited chemical characterization of the collected e-cigarette aerosols (i.e., nicotine quantification) (Table 2). FIGURE 3Open in figure viewerPowerPoint E-liquids, smoking machines, and e-cigarette devices (annotated within the figure) used in generation of aerosol(s) for in vitro research. ASPECG, aerosol single-port electronic cigarette generator; N/A, not available; NTV, novel tobacco vapor product; NVP, novel vapor product; ONARES, oro-nasal respiratory exposure system; SCSM, single cigarette smoking machine; UoK ASM, University of Kentucky analytical smoking machine FIGURE 4Open in figure viewerPowerPoint Number of puffs and vaping regimens used in the generation of aerosol(s) that were collected for in vitro research. CRM No. 81, CORESTA recommended method number 81; FTC, Federal Trade Commission; HCI, Health Canada Intensive; ISO, International Organization for Standardization 3308; N/A, not available; s, seconds; vol, volume 4 DISCUSSION Like in other areas of applied research, in vitro data have an important role to play in helping us comprehend the toxic potential of e-cigarette-derived aerosols in humans, while also supporting the 3Rs principles of scientific animal experimentation. Such data can help not only more readily identify hazards in an animal-cognizant manner but also potentially delineate modes-of-action (Ramirez et al., 2018; Shukla, Huang, Austin, & Xia, 2010); ultimately, this information will add to the weight-of-evidence that informs the risk assessment of e-cigarettes in relation to human health. Although sophisticated approaches involving three-dimensional organotypic respiratory tract cell cultures and whole aerosol exposure systems that partially recapitulate physiologically relevant exposure in humans might eventually become the key model for studying aerosol-associated toxicity, they are, at present, in their infancy and still require further exploration and validation (Bishop et al., 2019; Czekala et al., 2019; Iskandar et al., 2019; Mathis et al., 2013). Ostensibly until then, submerged two-dimensional cell culture-based assays will represent the core of in vitro assessments, in particular, for the internationally accepted tests that are used for identifying genotoxic and cytotoxic hazards, such the in vitro micronucleus, mouse lymphoma, bacterial mutagenicity, and neutral red uptake assays (INVITTOX, 1990; OECD, 1997; OECD, 2016a, 2016b). However, for the in vitro data to hold appreciable value in this context, it is vital that the captured aerosol is representative of its native aerosol. Significantly, underpinning this requirement is the collection method and solvent(s) employed. To this end, we surveyed the biomedical literature from the PubMed® repository for the approaches used by different laboratories across the world in their published in vitro research on collected e-cigarette-derived aerosols. Among the 47 relevant publications identified in the survey (including studies Breheny et al., 2017 and Rayner, Makena, Prasad, & Cormet-Boyaka, 2019 that described two methods each), 57% (28/49) of the collection methods were defined as "bubble-through." This is a method whereby aerosol generated from an e-cigarette is bubbled into a solvent, resulting in a solution containing the aerosol constituents, which can be subsequently applied to cell cultures. Furthermore, all 28 examples found in this survey employed water-based protic solvents—cell culture medium, FBS, PBS, or HEPES-buffered saline—and, thus, generated AQEs. It is, therefore, hypothesized that water-soluble constituents are captured predominately via this collection method, while other constituents—poorly and nonwater-soluble compounds for example—are probably not. Crucially, it should be noted that there is a disturbing lack of chemical characterization data on the collected aerosols among the studies in general; this is a critical finding, which we will address later in the manuscript. Nevertheless, there is empirical support for the theory of water-soluble constituent trapping via bubble-through/aqueous solvent-related methods from analytical studies on cigarette-derived GVP collected in PBS. These reports indicate that chemicals such as carbonyls, including acids, esters, amides, imides, aldehydes, and ketones, as well as lactones, alcohols, pyridine derivatives, imidazoles, lactams, and nitrogen heterocyclic compounds can be collected effectively by the bubble-through method (Noya et al., 2013; Schumacher, Green, Best, & Newell, 1977). In contrast, it is also recognized that numerous harmful and potentially harmful constituents from cigarette smoke (e.g., benzo[a]pyrene, dibenzo[a,h]pyrene, and 5-methylchrysene) are highly lipophilic (i.e., possessing octanol–water partition coefficients [log P] > 5) (Smith & Hansch, 2000). Thus, if these types of molecules are present in the aerosols produced from e-cigarettes, they are most likely not captured by aqueous solvent-centric methods because of their inherent chemical properties. The next most frequently employed collection method was CFP (18%; 9/49). In this method, e-cigarette-derived aerosol is pulled through a CFP to capture its constituents on the filter pad (i.e., ACM). These constituents are subsequently desorbed and solubilized in a solvent. These nine publications employed different polar solvents, both protic (cell culture medium and PBS) and aprotic (DMSO) in nature, which yielded fractions that were defined as AQ- or DMSO-soluble ACM. Importantly, total particulate matter fractionated from cigarette smoke has been extensively characterized owing to the virtues of CFP-mediated collection (Chepiga et al., 2000; Roemer et al., 2004). Thus, while the same kind of particulate matter (carbon-based) is not present in the aerosol of e-cigarettes because of the absence of combustion (Lampos et al., 2019), one might expect that the approach has the potential to capture a similar profile of chemicals, although the adherence capacity of aerosol components towards the CFP as well as their solubility in the applied solvent will obviously dictate which constituents finally comprise the fraction. Predicated upon published examples, this approach can potentially trap chemicals such as nicotine, glycerol, aromatic amines, and polycyclic aromatic hydrocarbons (Chepiga et al., 2000; Roemer et al., 2004). Interestingly, a recent publication reported that a CFP method is more effective in collecting a targeted set of flavor chemicals than its bubble-through counterpart, indicating that this sorbent-based technique might have advantages over others (Eddingsaas et al., 2018). However, one possible limitation of this capture method is that aerosol constituents not retained by the CFP are, presumably, poorly collected. The tandem combination of CFP and bubble-through methodologies (defined as "CFP + bubble-through") was cited once in this selection of curated literature (2%; 1/49). DMSO was used to solubilize and elute ACM from the CFP, while PBS was used to capture a portion of the constituents passing through the CFP; thus, it is anticipated that the two fractions contained polar/nonpolar and non-CFP immobilized water-soluble constituents, respectively. When aerosol is collected in this manner, toxicological assessment of both fractions—as was done in previous in vitro assessments of cigarettes and heated tobacco products (Gonzalez-Suarez et al., 2016; Rickert, Trivedi, Momin, Wright, & Lauterbach, 2007; Roemer et al., 2015; Schaller et al., 2016)—might provide a better understanding of the hazard potential of the aerosol in its entirety. The "condensation" collection method was cited five times (10%; 5/49), and, in four of the five studies, fractions defined as condensates were produced and then assessed in vitro. While this approach has potential advantages (e.g., circumventing the need for a collection sorbent or solvent), it is not clear which, and at what proportion, aerosol constituents (other than nicotine) are condensed and, therefore, present in the final fraction. In the fifth study, the condensate was subsequently solubilized in a solvent syste