Table 1.

Comparisons summary of 2D culture and 3D culture.

	Models
	2D Culture	3D Culture		References
	Cell Culture	Spheroids	Organoids
Accessibility	More easily accessible as well-established culture reagents and cell lines easily available.	Accessible due to cellular sources and reagent availability.	More novel than the previous two methods, accessibility remains limited due to specific reagent suppliers and tissue availability from patients.	(59)
Cost to maintain cultures	Relatively cheap to maintain as commercially available reagents and assays.	Lower cost in comparison to organoids, but requires time to initiate and develop cultures.	More expensive, fewer commercially available tests and reagents. Can also be quite costly to initiate and develop cultures.	(58, 60)
Well-established culture protocols	Well-established and standardized protocols available for a variety of cell lines.	There are a number of well-established protocols (suspension culture, scaffold culture, etc) but use may be dependent on the type of experiments	Some well-established protocols available but no standardized protocols.	(56, 59, 61)
Time expenditure to establish culture	Within a few hours to a few days	Within a few days to a few weeks	Within a few weeks to a few months	(62–64)
Patient-specific & cellular sources	Yes, can derived from an established single cell-line or from patient primary cells	Yes, can be derived from patient primary cells or established cell-lines can be used	Yes, patient samples can be digested and specific cell-types harvested to generate organoids	(16)
Cellular characteristics	Cells often present with a flat and elongated shape. Cells are grown as monolayers and can only expand in two-dimensions	Cells are able to maintain their natural 3D shape and are able to form multiple layers and grow in three-dimensions as it would occur in-vivo		(60, 65–68)
Cell proliferation rates	Cell proliferation occurs at an unnatural rate and often cells are at the same stage of growth	Cell proliferation occurs at more natural rates and cells are sometimes at different stages of growth (as seen in-vivo)		(67, 69)
Gene and protein expression levels	Expression levels are vastly different when compared to in-vivo systems	Expression levels have been found to resemble those seen in-vivo		(60, 66, 67, 69)
Sensitivity to drugs	Cells present with minimal resistance to drugs, which can result in successful outcomes to administered drugs	Often present with more resistance to administered drug treatments, showing a more holistic overview to drug metabolism in-vivo.		(67, 70, 71)
Tumor microenvironment	Single cell types are often cultured without additional cell types, there is limited tumor heterogeneity, there is often no ECM substance and cell-cell interactions is limited to between one cell type. Therefore, does not accurately represent the tumor microenvironment.	Multiple cell types can be cultured together, there is better tumor heterogeneity observed, ECM substrate is present and cell-cell interactions can be observed between different cell types. Therefore, the tumor microenvironment is more accurately represented.		(72, 73)
Usage, analysis & reproducibility	Easily used to generate results and analysis is easily interpretable. Results are also easily reproducible	Usage can vary from user-to-user and from experiments, analysis can also be difficult to perform due to 3D nature and reproducibility remains an issue		(56, 74, 75)