Table 2.

Main methods used to generate spheroids.

Method	Procedure	Matrix	Advantages	Disadvantages	Ref.
Liquid overlay	Tumor cells are placed on tissue culture plastic covered with a thin layer of inert substrate.	Agar Agarose PolyHEMA	Easy-to-use protocol; Easily promotes the aggregation of cells to become spheroids; Co-culture ability; High reproducibility; Inexpensive; Easy to image.	Difficulty to monitor the number and size of spheroids; Heterogeneity of the cell lineage; Lack of interactions between cells and matrix.	[7,34,47]
Ultra–low attachment plates	Cells are seeded in an ultra-low attachment plate without coating as the polystyrene surface offers low adhesion properties.	-	Capability to produce one spheroid per well; Spheroids have a more compact structure than those on agar-coated plates; Easy to image.	Difficulty to monitor the number and size of the spheroids; Heterogeneity of the cell lineage; Lack of interactions between cells and matrix	[48]
Hanging drop method	Cells are dropped in a small volume in the petri dish lid. The lid is subsequently inverted, and aliquots of cell suspension turned into hanging drops without dripping due to surface tension.	-	Easy-to-use protocol; Consistent size and shape controlled by adjusting the density of cell seeding; High reproducibility; Inexpensive; Easy to image.	Heterogeneity of cell lineage; Lack of interactions between cells and matrix; Limited volume of the cell suspension; Difficulty in changing the culture medium.	[49]
Hydrogel embedding/Scaffold	Microcapsules with matrix /cells obtained from cells resuspended in hydrogel 3D structures that are constructed from a wide-range of materials and possess different porosities, permeabilities, surface chemistries, and mechanical characteristics.	Alginate Matrigel Methylcellulose Collagen Gelatin Silk Chitosan	Large variety of natural or synthetic materials; Customizable; Co-culture possible; Resemble natural extracellular matrix; Circulation of nutrients and cellular waste in and out of the hydrogels.	Deficiency in gelation kinetic control; Undefined composition in natural gels; May not be transparent; Difficulty to remove cells.	[50,51]
Spinner flask bioreactor	Cells are inserted into a chamber with continuous agitation (by gently stirring, rotating the chamber, or perfusing culture media through a scaffold using a pump system). Bioreactors are equipped with media-flowing systems to provide nutrient circulation, metabolic waste removal, and homogeneity of the physical and chemical factors within the bioreactors.	With or without matrix	Easy-to-use protocol; Great spheroid formation; Precise control system and guaranteed reproducibility; Motion of culture assists in nutrient transport; Large scale production.	No control of the cell number/size of spheroids; Cells possibly exposed to shear force in spinner flasks; Specialized equipment required.	[52]