Table 3.

Overview of spheroid and organoid formation methods

Method	3D culture system	Description	Advantages	Challenges	Refs.
ECM scaffolding	Organoids	Stem cells are placed in Matrigel (or ECM mix) and maintained in culture	Replicates microenvironment Observation of cell adhesion and migration	Lack of reproducibility using natural ECM Synthetic ECM requires upregulating reagents	[ 117 , 125 , 126 ]
Hanging drop	Spheroids	Cells are suspended in media droplet. Cell aggregation occurs at the air–liquid interface	Consistent Does not require ECM Possible to integrate array production	Difficulties with media change Small size Low throughput	[ 127 , 128 ]
Low‐adherent surfaces	Spheroids	Cells are suspended and cultured on a low‐adherent plate, or hydrophilic substrates (i.e., hydrogel) to form aggregates	Does not require ECM Cost‐effective	Not adaptable to all cell types Heterogeneous population	[ 129 ]
Spinning bioreactor	Spheroids and organoids	Cell suspension is housed inside a spinner flask or a bioreactor with continuous mixing. Cell aggregation is induced by convectional force	Can generate a wide range of model sizes	Large and heterogenous structures Shear forces on cells	[ 130 , 131 ]
Magnetic levitation	Spheroids and organoids	Nanoparticles are ingested by cells, which are then placed in a low‐adherent substrate. A magnet lid is used to induce cell aggregation	Does not require ECM or media	NPs are expensive and toxic	[ 132 ]
Bioprinting	Organoids	Additive manufacture of cytokines, cells, and ECM	Generates complex and organized structures Use of multiple cell types	Bioink selection with desired characteristics	[ 133 , 134 , 135 ]
Micropatterning	Spheroids	Microcontact printing and soft‐lithography patterning of ECM	Structure control Array production	Expensive equipment Poor reproducibility Lack of patterning efficiency	[ 136 ]
Microfluidics	Spheroids and organoids	3D structures housed inside microstructures, integrated with microsensors	Replicates microenvironment Allows for nutrient delivery Avoids necrosis Array production	Low cell recovery Postcell analysis challenges	[ 137 ]