Table 2.

Advantages and disadvantages of 3D cell culture protocols.

Methods		Advantages	Disadvantages	References
Synthetic scaffolds	Metals	Biocompatible Great mechanical properties	Potential poor biodegradability Oxidation and aggregation issues May require to be combined with a polymer Secondary release of metal ions may cause toxicity	[101]
	Ceramics	Osteoconductive and osteoinductive properties (bioactive ceramics) Composition can be similar to the human bone mineral content	Significant brittleness May display inappropriate degradation/resorption rates	[102]
	Polymers	Good tunability of physical properties Low immune response Low production cost High reproducibility Defined purity and composition	Often hydrophobic Lack of cellular recognition patterns for some of them Poor biocompatibility Risk of biodegradation side effects (inflammation, toxicity, etc.)	[101,102]
Natural scaffolds	Polysaccharides	High biocompatible Low toxicity Biodegradable Often contain biofunctional molecules, cell recognition patterns, and adhesion sites on their surface Similarity with native ECM	Limited physical properties Might contain pathological impurities such as endotoxin Difficult to process Properties dependent on extraction and processing procedures	[101,102]
	Proteins	Biocompatible Biodegradable Similarity with native ECM Good interactions with the cells	Limited physical properties Low stability Possible transfer of pathogens Composition varies between batches Unidentified growth factors and bioactive components	[102,103]
	ECM-derived	Natural ECM components Good interactions with the cells Contain biofunctional molecules Minimal processing	Uncertain composition In-between lots variations Risk of unwanted interactions or interferences in signaling pathways	[104]
	Acellular matrix	Preservation of the native ECM High biocompatibility	Incomplete decellularization may generate unwanted immune and inflammatory responses	[105]
Hydrogels	Natural/ Synthetic	High water content Can be made from a large variety of natural or synthetic materials Highly biocompatible Controlled degradation rate Highly tunable Inexpensive Co-culture possible High reproducibility	Limited physical properties Gel-to-gel variations Structural changes over time	[105,106]
Scaffold-free	Spheroids	Simple protocols Highly reproducible Possibility to use multiple cell types Size is easily tunable Inexpensive	Simplified architecture Limited flexibility Limited size Lack of porosity Lack of matrix interaction	[106,107]
	Organoids	Cells can be extracted from a patient Great biomimicking abilities	Has a certain variability Hard to reach in vivo maturity Lack vascularization Can lack key cells types	[107]
	Self-assembly	Cells produce their own ECM Cells can be extracted from a patient Enable the formation of tissue-mimicking organ-specific tissue depending on the cell types used	Requires long culture time Limited size Limited mechanical properties Lack vascularization Incomplete cell differentiation alters biomimicking properties.	[108]