Table 1.

Methods for spheroid formation

Method	How to	Driving force	Advantages	Disadvantages
Hanging drop	Make droplets of cell suspensions on a lid of tissue culture plate, the lid is flipped upside-down, and culture in a humid condition	Gravitational forces and physical confinement	Easy to control spheroid size	A limited volume of droplets (<50 μl) Difficulty in changing culture medium
Microwells	Confine cells in physical compartments at a micrometer scale	Gravitational forces and physical confinement		A difficulty in harvest
Centrifugation	Force cells aggregate at the bottom of a centrifuge tube	Centrifugal forces		Cellular damages by excessive external forces
Magnetic levitation	Force magnetized cells form into spheroids	Magnetic force		Cytotoxicity of magnetic materials
Microencapsulation	Confine cells into microcapsules	Physical confinement		Batch variations
Non-adherent plates	Interrupt cell adhesion to the plates, make cells rather aggregate to each other	Spontaneous aggregation	One step spheroid formation and suspension culture Less labor intensive Suitable for large scale spheroid formation	Low yield and various spheroid size
Rotating wall vessels	Create a microgravity environment	Shear force
Spinner flasks	Generate dynamic fluid shear force	Shear force