Table 1 Summary comparison of 2D and 3D methods, advantages and disadvantages

Techniques

Cells are cultured on flat, adherent surfaces, typically made of plastic or glass, and usually coated with substrates (e.g. laminin, PDL) to enhance cell adhesion and/or direct differentiation

Scaffold-based systems based on a solid or liquid matrix of either natural or synthetic material (e.g. inert electrospun scaffolds, natural and synthetic hydrogels). Cells are typically seeded onto/into scaffold materials

Scaffold-free systems (e.g. self-assembled spheroids, organoids or cell aggregates).

Advantages

Simplicity of use (e.g. for less experienced users and typically not requiring specialist equipment)

Inexpensive

Homogenous culture

Reproducible

Well-established technique (e.g. for comparison with existing data)

Ease of access to cells for downstream applications and for visualisation techniques (e.g. microscopy)

Allow more complex interactions between cells

Allow cell-ECM interaction

Can provide better spatial organization

Higher degree of complexity for more relevant models of in vivo environment and tissues

Scaffold-based systems can be designed to provide specific chemical and physical cues (e.g. functionalisation, changes in pore size and stiffness)

Disadvantages

Not a good representation of the in vivo, physiological environment

Cell-cell interaction largely limited to side-by-side contact

Lack of predictive ability for in vivo events

Lack of relevant cell-ECM interactions

Results in cell flattening/altered morphology

Leads to altered gene expression

Can be expensive (particularly in comparison to 2D)

Can present a greater challenge for visualisation/microscopy techniques and other parallel methodologies (e.g. patch clamp electrophysiology)

Can be challenging for homogeneous distribution of components (e.g. oxygen and nutrients), leading to necrotic areas, cell death or heterogeneity

May require specialised and expensive equipment (e.g. bioreactors) and expert handling and optimisation

Potential for reduced reproducibility, including variability of natural scaffold materials

Scaffold-based approaches must take into consideration material properties (e.g. biodegradability, pore size, chemical composition)

Scaffold-based platforms can increase the difficulty of retrieving cells for downstream applications