Refining in vitro models for nanomaterial exposure to cells and tissues

With the increasing use of nanomaterials (NMs) in a variety of commercial and medical applications, there is a parallel increase in concern related to unintentional exposure. This leads to a pressing need for appropriate hazard and risk assessment, and subsequent regulation of these new and emerging nanosubstances. Typically, in vitro models are the first point for assessment, and these are often then used to begin to predict and translate the potential effects in vivo. The area of nanotoxicology is therefore critically important, and requires that experimental protocols are clear, defined and standardized within adequate risk assessment frameworks to allow hazard identification and extrapolation to more realistic in vivo situations. Often, however, results seen in vitro do not adequately represent the situation in vivo. There are many differences between in vitro and in vivo investigations; however one issue may arise from the way in which typical nanosafety studies are carried out. Nanotoxicity assessment requires a definition of dose and standardized modes of exposure in nanomaterial uptake and cytotoxicity studies to determine the hazard that is posed by naturally occurring and engineered (E-)NM. Current methods in nanotoxicity studies often report only the exposure dose, which can lead to large variations in the intrinsic or delivered dose due to inhomogeneous exposure and loss of treatment material depending on how NMs are “presented” to cells. Protocols for NM dispersion and cellular assay design therefore require Standard Operation Procedures (SOPs). Many experimental conditions in NM studies directly affect the NM behaviour within the cell culture system, in particular handling of NM dispersions as well as the order, timing and exposure configurations of the incubation; these can have an immediate effect on the resulting physical distribution of NMs on the cell surfaces. Here we review handling, physical conditions and particle distribution in cell models for NM exposure to cells and tissues. Efforts to improve in vitro models such that they more closely mimic the in vivo conditions, such as exposure mechanism and dose, and potential for transport of NMs across cells are being developed rapidly, driven partially by the strong push within the EU to reduce animal testing. Examples include the use of multiple in vitro assays to calculate hazard, air-liquid interface (ALI) exposure, microfluidic approaches and 3D co-culture models.