Table II:

Advantages and challenges of three-dimensional (3D) tumor models.

Techniques	Advantages	Challenges	Research Stage	Ref
Liquid Overlay Culture and Hanging Drop	Simple to use Uniform spheroid formation Co-culture feasibility Control cell size Transparent Inexpensive High reproducibility Ease of harvesting Long term culture	Limited flexibility Lack reproducibility Lack cell-matrix interaction Heterogeneous cell lineage spheroids Low throughput Spheroid handling and transfer critical	Basic Research Drug Discovery Personalized Medicine	[210, 220, 221]
Hydrogel	Inexpensive Uniform sized microtumors Co-culture feasibility High reproducibility Customizable microtumor size Cell-ECM interactions	Gel Composition Gel-to-gel variations and structural changes Non-Transparent Gelling Mechanism Handling microtumors Isolation of specific microtumors Low throughput	Basic Research Drug Discovery	[222–226]
Bioreactors	Simple to culture High-throughput production Easily accessible spheroids Motion of culture assist nutrient transport	Specialized equipment required Cell Number/size cannot be controlled Cells exposed to shear force	Basic Research Tissue Engineering Cell Expansion	[161–163, 227–233]
Scaffolds	Medium cost Co-culture feasibility Customizable	Scaffold-to scaffold variation Not transparent Difficulty in cell removal	Basic Research Drug Screening Cell Expansion Drug Discovery	[157, 234–237]
Microtitre plate	Controlled spheroid size Cell growth and assays in same well HTS/HCS compatible	Limited work on ECMs	Basic Research Tissue Engineering Anticancer Drug Screening	[132]
Microfluidics	Chemical gradients Cell localization studies Transparent Co-culture feasibility	Fluid control Environment control Require expertise High microfabrication cost	Basic Research Tissue Engineering	[238–240]
3D Bioprinting	Chemical and physical gradients Co-culture feasibility Customized architecture High-throughput production	Expensive 3D bioprinting machine required Challenges of cell/material used	Cancer Pathology Anticancer Drug Screening Cancer Treatment Tissue Engineering	[165, 168, 169, 241]

Extracellular matrix (ECM), High-throughput setting (HTS), High content screening (HCS).