Table 5 Overview of the main in vitro clonogenic assay models

Traditional 2D Monolayer Colony Formation.

Low cost;

Easy and rapid seeding and colony counting.

Difficult to translate results to in vivo due to monolayer growth;

The clonogenic potential may be different by adherent growth.

Franken et al. (2006); Rafehi et al. (2011)

Spheroid-Based Colony Formation.

Similarity to tissue architecture in vivo;

Suitable for testing NP penetration into tissue.

Results depend on spheroid morphology (loose vs compact morphology);

Complexing counting procedure since extracellular matrix can hinder image quality;

More time is required.

Franken et al. (2006); Chermat et al. (2022)

On-a-Chip Colony Formation.

Architecture can mimic physical microenvironments with accuracy;

Good image quality and easy colony count;

Can assess treatment effects in dynamic flow.

High cost;

Preparation and analysis can be laborious.

Patra et al. (2019); Brix et al. (2021)