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The intrinsic limitations and pitfalls exhibited by two-dimensional (2D) in vitro studies in conjunction with the ethical issues surrounding in vivo (nano)toxicology testing on animals have prompted the need to develop three-dimensional (3D) models. This chapter discusses the problems faced when using monolayer cell cultures for safety assessment, which led to the rationale behind the development of 3D models for safety evaluation purposes. The additional benefits of 3D models for nanosafety are presented, coupled with an overview of some of the 3D in vitro models that have been developed to mimic the physiological environment of certain tissues/organs including liver, lung, colon, intestine and gastrointestinal tract.

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