AnIn-VitroStandardized Protocol for Preparing Smoke Extract Media from Cigarette, Electronic Cigarette and Waterpipe

Cigarette smoke exposure is a major risk factor for various diseases, and in vitro studies using smoke medium (SM) are crucial for elucidating underlying mechanisms. However, the lack of standardized protocols for SM preparation has led to inconsistencies in experimental results. This paper presents an optimized protocol for reproducible generation of SM from conventional cigarette smoke extract (CSE), electronic cigarette smoke (ECS), and waterpipe vapour (WPV). The protocol involves: A custom-built apparatus for smoke capture in a cell culture medium Specific parameters mimicking human smoking topography Rigorous aseptic techniques and safety measures Extensive details are provided for material preparation, apparatus setup, smoke generation procedures, and post-processing steps. This standardized method enables reliable in vitro exposure models, easing comparative studies across different tobacco products. To prove protocol consistency, we present optical. Additionally, we highlight the protocol’s utility by figuring out the SM quality through analysing O.D. data and morphological changes, cell viability assays, ELISA and gene expression analyses i.e., RNA sequencing through several in-vitro models, i.e., Human Umbilical Vein Endothelial Cells, (HUVECs, Merck) , human leukaemia monocytic cell line (THP-1, Sigma) and induced pluripotent stem cells differentiated to cardiomyocytes ( iPSC-CMs, Axol) respectively. This optimized protocol addresses variability in SM preparation, enabling consistent investigations into smoke-induced pathologies and potential therapeutic interventions. Graphical abstract Specifications table