Cells in a 3D environment are good models as \"near-to-in vivo\" systems and give us useful insights from a variety of aspects [17]. They can serve as a cost-effective platform to provide a better and more realistic predictive value for safety and risk assessment. Therefore, 3D cell culture platforms that could better recapitulate the in vivo tissue functions that have been developed. Many biocompatible 3D scaffolds have been developed as 3D cell culture systems, especially for the application in tissue engineering. These scaffolds are structured to mimic the characteristics of ECM to reflect in vivo conditions, helping the cells to maintain a more representative structure, gene expression and functions. Cells could grow layers on the surface and inside the porous scaffolds. One of the extremely relevant approaches is hydrogel system that created from the natural or synthetic polymers [18; 19; 20]. Cells are encapsulated in soft gels to grow and form more physiologically relevant 3D constructs. 3D printed scaffolds [21; 22] and porous polymeric microspheres based on the biodegradable polymers [23; 24], such as poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone, could also serve as a 3D cell culture platform. 3D structure meshes based on electrospun fiber represent another typical platform for 3D cell culture, in which cells grow in and around the fibrous 3D topography [25]. Amongst the varied scaffolds applied for 3D cell culture, porous polymers derived from high internal phase emulsions (polyHIPE) have been widely investigated due to their high porosity and controllable morphology that benefit the cell growth [26; 27; 28; 29; 30; 31; 32; 33; 34; 35]. For example, biodegradable porous scaffolds, such as PLGA [33], polycaprolactone (PCL) [34], gelatin [35], were prepared by the high internal phase emulsion templating method and demonstrated great potential for tissue engineering. Furthermore, polystyrene-based polyHIPE materials [36; 37], which have a similar chemical composition to conventional tissue culture plate and appropriate mechanical properties for routine handling, were also demonstrated to be suitable for 3D cell culture applications. For the fabrication of polystyrene-based polyHIPE, a water-in-oil (W/O) high internal phase emulsion (HIPE) was prepared, followed by the polymerization of the external phase which contains polymerizable monomers, such as styrene (St) and divinylbenzene (DVB). The internal water phase acts as the porogen and is removed after polymerization of the HIPE, resulting in spherical macropores (named \"voids\", typically up to 10 \(\upmu\)m in size). The manipulation of the initial emulsion would help to control the void size for excellent scaffold reproducibility and cell growth. Each void is interconnected with small holes (named \"windows\", usually 1-2 \(\upmu\)m in size), which are resulted from the contracting and rupturing of the thin external phase film during polymerization [38]. The porous PolyHIPE materials have high porosity and a large number of spherical voids that are highly connected with small window throats, which would facilitate nutrient and waste transfer for cells growing in the materials. These 3D platforms have been demonstrated as useful for 3D cell growing, with the great application prospects in the field of tissue engineering, regenerative medicine and drug discovery. However, the application of such 3D cell culture platforms in the field of cytotoxicity evaluation of cigarette smoke is rarely reported. In this paper, we aimed to construct an easily fabricated 3D cell cytotoxicity evaluation system based on the porous scaffolds. Due to their high porosity, open-cell structure with connected windows, and feasible fabrication, polystyrene-based polyHIPE materials were chosen as scaffolds. The polystyrene-based polyHIPE materials were prepared by the copolymerization of St and DVB in the continuous phase of a HIPE.