Table 3.

Summary of main advantages, limitations, and applications of cell culture strategies for nanomedicine in vitro evaluation.

Strategy	Main advantages	Main limitations	Highlighted applications	Examples with nanomedicine
Monolayer cell culture	Inexpensive	Cell-line derived: clonal selection	Initial nanomedicine biological characterization.	[67]
Co-culture	Representation of different TME populations	Condition optimization for the growth of different cell lines	Cell type-targeted therapies	[69], [95]
Spheroid	Representation of: Cell-cell interactions Cell-matrix interactions In vivo-like gradients (over 400 µm diameter) Do not require highly specialized techniques	Cell line-derived: clonal selection Higher costs than monolayer culture	Tumor penetration studies	[75], [94], [95]
Organoid	Patient-derived Maintenance of intra- and inter- tumor heterogeneity	High costs Complex establishment and maintenance compared to cell line-derived cultures	Patient-tailored therapies	[87]
Tumor-on-chip	Supports microfluidics	High complexity Specific expertise and materials required	High-throughput screening	[98]
Bioprinting	High spatial control	Specific expertise and materials required	Tissue and organ fabrication when patterning and precisely biologics placing is required	[103], [104]

Tumor microenvironment: TME