Table 2.

Overview of in vitro 3D models.

Methods	Size	Culture Time	Advantages	Disadvantages	Ref
Scaffold-based approach	Scaffold dependent, up to a few mm	Scaffold degradation dependent (1 day to several months)	Cell adhesion, proliferation, differentiation depending on scaffold physicochemical properties Oxygen and nutrients transport depending on scaffold permeability High reproducibility ECM organization control	Cellular adhesion and growth changes depending on scaffold material Cell behavior changes depending on cellular topography distribution	[6,45,46,81,82,83,84,85,86]
Spheroids	<1 mm	Up to 2 months	Reproducible Nutrients and oxygen gradients Optimal cell–cell and cell–ECM interactions Long term culture (≈2 months)	Simplified architecture Hypoxic center Self-renewal deficit	[45,87,88,89]
Low-adhesion plate/ Non-adherent surface			Easy to use All steps in the same plate High throughput	Heterogeneous size Find an appropriated cell ratio for co-culture Spheroid formation with few cells	[34,90,91,92]
Hanging drop			No scaffold needed Homogeneous size Low number of cells required Co-culture control	Low throughput Need to change plates for assays Time consuming Challenging media renewal methods Difficult long term culture	[86,89]
Hanging drop plate			High throughput Homogeneous size Low number of cells required Co-culture control	Challenging media renewal methods	[88,90,93]
Scaffold-based method			Mimic in vivo environment Cell–ECM interactions High throughput	Scaffold changes between batches Natural hydrogels: weak mechanical properties, rapid degradation Synthetic scaffold: biocompatibility issues	[66,85,86,94]
Suspension cultures: spinner flasks/bioreactor			Cell–cell interactions High throughput Mass production	Specialized equipment High shear forces (bioreactor < spinner flask) Heterogeneous size and composition	[90,95,96,97,98]
Magnetic levitation			Fast spheroid formation ECM intrinsic formation	Expensive beads Limited cell number	[90,99,100]
Organoids	0.5–4 mm	Up to 1 year	Long term culture (≈1 year) Spontaneous formation Mimics embryonic development Cell self-organization Complex tissue organizational capacities like in vivo	Heterogeneous shape and size Low reproducibility Hypoxic centers Lack of key cell types (most of time) Instability between batches	[45,101,102,103]
Microfluidic System	<1 mm	Scaffold degradation dependent (1 day to several months)	Spatio–temporal environment control Dynamic culture Co-culture Cell-patterning control	Challenging technical side (microcircuits) Specialized equipment (pump, device) Challenging media renewal methods Different culture surfaces	[45,72,92,104,105,106]
3D Bioprinting	Scaffold dependent, up to a few cm	Scaffold degradation dependent (1 day to several months)	Robotized Cell-patterning control ECM configuration control Possible combination of 3D models	Expensive (bioink, printers) Possible collapse of layers	[45,107,108,109,110]
Inkjet			High speed Low cost >85% cell viability	Low precision Few cells, low viscosity	[111,112,113,114,115]
Microextrusion			Easy to use High cell density and high viscosity bioink	Low cell survival: 40% viability Cell structure distortion	[18,111,116,117]
Laser-assisted			High cell density and high viscosity bioink High precision 95% cell survival	High cost Time-consuming ribbon manufacturing	[109,111,118]
Stereo-lithography			High precision >90% cell viability	Strong UV light use Polymer biocompatibility and biodegradability	[111,119,120,121]