In vitro models are essential tools for evaluating the potential toxicity of nanomaterials, offering controlled, reproducible, and ethically favorable alternatives to in vivo studies. These models range from traditional 2D cell cultures to advanced 3D systems and organ-on-a-chip platforms. Each approach has distinct advantages, limitations, and standardization challenges that influence their applicability in nanotoxicology.

**2D Cell Cultures**
Monolayer cell cultures are the most widely used in vitro models for nanotoxicity assessment due to their simplicity, cost-effectiveness, and ease of handling. Commonly employed cell lines include:

- **A549 (human lung adenocarcinoma)**: Used to study pulmonary toxicity, particularly for inhaled nanoparticles. These cells mimic alveolar epithelial responses and are sensitive to oxidative stress induced by metal oxides like TiO2 and ZnO.
- **HepG2 (human hepatocellular carcinoma)**: A liver model for evaluating nanoparticle metabolism, detoxification, and hepatotoxicity. HepG2 cells express phase I and II enzymes, making them suitable for studying nanoparticle-induced liver damage.
- **THP-1 (human monocytic leukemia)**: Differentiated into macrophage-like cells to assess immune responses, including cytokine release and phagocytosis of nanoparticles.
- **Caco-2 (human colorectal adenocarcinoma)**: Used for intestinal barrier studies, particularly for ingested nanomaterials. These cells form tight junctions and mimic the intestinal epithelium.

**Advantages**: High throughput, low cost, and well-established protocols.
**Limitations**: Lack of tissue complexity, altered cell behavior due to artificial surfaces, and absence of systemic interactions.

**3D Cell Cultures**
3D models better replicate the structural and functional complexity of tissues compared to 2D cultures. Common types include spheroids, organoids, and scaffold-based systems.

- **Spheroids**: Self-assembled aggregates of cells (e.g., tumor spheroids) that mimic cell-cell interactions and nutrient gradients. Used to study nanoparticle penetration and localized toxicity.
- **Organoids**: Miniaturized organ-like structures derived from stem cells, offering patient-specific toxicity data. Liver organoids, for example, provide insights into nanoparticle metabolism.
- **Scaffold-based cultures**: Synthetic or natural matrices (e.g., collagen, hydrogels) that support 3D cell growth. These models are useful for assessing nanoparticle effects on extracellular matrix interactions.

**Advantages**: Better physiological relevance, inclusion of cell-matrix interactions, and improved prediction of in vivo outcomes.
**Limitations**: Higher cost, technical complexity, and variability in size and composition.

**Organ-on-a-Chip Systems**
Microfluidic devices that simulate organ-level functions by culturing cells in dynamic, mechanically active environments. These systems integrate fluid flow, mechanical forces, and multi-cell interactions.

- **Lung-on-a-chip**: Models the alveolar-capillary interface to study nanoparticle deposition, clearance, and inflammatory responses.
- **Liver-on-a-chip**: Incorporates hepatocytes and non-parenchymal cells under perfusion, enabling real-time analysis of nanoparticle metabolism and toxicity.
- **Gut-on-a-chip**: Replicates peristalsis and villi structures to assess nanoparticle absorption and barrier disruption.

**Advantages**: High physiological fidelity, ability to study systemic interactions, and real-time monitoring.
**Limitations**: High fabrication costs, operational complexity, and limited throughput.

**Standardization Challenges**
Despite their utility, in vitro nanotoxicity models face several standardization issues:

- **Dosimetry**: Nanoparticle agglomeration, sedimentation, and diffusion rates vary across models, complicating dose comparisons.
- **Characterization**: Inconsistent nanoparticle characterization (size, surface charge, purity) leads to reproducibility issues.
- **Endpoint selection**: Variability in cytotoxicity assays (e.g., MTT, LDH, ROS measurements) affects data comparability.
- **Biological relevance**: Many models lack immune, endocrine, or neural components that influence nanoparticle toxicity in vivo.

**Conclusion**
In vitro models for nanotoxicity assessment span from simple 2D cultures to sophisticated organ-on-a-chip systems. While 2D cultures remain the workhorse for high-throughput screening, 3D and organ-on-a-chip models offer superior physiological relevance. However, standardization of dosimetry, characterization, and endpoints is critical to improve reproducibility and predictive accuracy. Future advancements should focus on integrating multiple organ systems and refining microphysiological conditions to bridge the gap between in vitro and in vivo outcomes.