Table 1.

The advantages and drawbacks of the various in vitro methods.

Type	Advantages	Drawbacks	References
2D cell culture
Upright NM exposure	Easy experiment set-up; Can be used for virtually all 2D cell cultures;	Agglomeration of nanoparticles; Inconsistent protocols between studies; Inhomogeneous distribution over time	[78]
Surface-based NM presentation	Exact NM/µm; No agglomeration of particles; Homogeneous distribution over time Easy monitoring of uptake and toxicity	NM–substrate interactions influence internalization and toxicity results; Only static conditions can be tested.	[99]
Inverted cell culture	Assessment of buoyant NM nanotoxicity	Limited use for larger-sized or insoluble NMs	[79,80,81,82]
Air liquid interface	More physiologically relevant; Cheaper than in vivo studies; Range of commercially devices available	Limited to airborne NMs; Only relevant to nanotoxicity studies related to inhalation	[83,85]
3D cell culture
Co-culture	Promotes in vivo-like cell–cell interactions; More relevant than 2D nanotoxicity platforms;	Still lacks 3D microenvironment	[86]
Spheroids and organoids	More in vivo-like complexity; Oxygen and nutrient gradient; Barrier to NMs distribution and nanotoxicity; Easy-to-use protocols	Heterogeneity; Lower reproducibility; Simplified 3D architecture; No high throughput	[86,87,89,90]
Organ-on-Chip	High throughput; Low cost; Physiologically relevant microenvironment; Precise control over NM presentation and dosimetry	Surface effects stemming from small dimensions; Little mixing of solutions; Difficult integration of sensors;	[91,92]
Precision-cut tissue slices	Compatible with a range of tissue samples and animal species; High reproducibility; Quickly obtainable; Retain the tissue native architecture	Tissue damage due to slicing; Limited number of slices per organ	[93,94,95,96,97]