Table 4.

Advantages and disadvantages of the different techniques employed to generate spheroids.

Techniques	Advantages	Disadvantages	Applications
Matrix on-top and matrix-embedded	Cells can be recovered post-culture if self-aggregating protein based hydrogel is used.	Hydrogel require special handling. Yields heterogenous spheroids requiring sorting before assay. Challenging to stain and image matrix-embedded spheroids.	Ideal for evaluation of cell-cell and cell-matrix interactions, drug screening, cancer cell metabolism. Allow hypoxia related studies. Allow evaluation of cancer stem cell niche.
Matrix encapsulation (microfluidic device)	Yields homogenous spheroids circumventing the need for sorting before assay.	Slower growth rate due to confinement. Increased occurrence of necrosis due to confinement. Capsule may burst if the matrix shell is thin.
Micropatterned plates	Spheroids can be imaged with relative ease. Post culture recovery is possible ECM component is present.	Well surface needs to be coated tp create low adhesion surface. Generates spheroids of variable sizes. Multiple spheroids in a well can overwhelm assay chemistry.
Hanging drop	Large number of spheroids obtained in a limited space. Reduced reagent consumption. Post culture recovery is possible.	Labor-intensive. For long term culturing, spheroids are transferred from the hanging drop to a second plate that can hold larger volume of media. Spheroids are transferred to a secondary plate for end-point analysis.	Ideal for studying invasive potential of cancer cells. Allow evaluation of cancer stem cell niche. Ideal for drug screening.
Ultra low attachment plates	Inexpensive and easy to handle. Large number of spheroids can be obtained in a limited space (96 well or 384 well). End-point analysis can be done on the same plate. Post culture recovery is easy. Can be multiplexed with imaging and other biochemical assays.	Generates spheroids of variable sizes. May have a mixture of attached cells and spheroids
Magnetic levitation and Magnetic Bio-printing	End point analysis can be done on the same plate. Can be multiplexed with imaging and other biochemical assays.	Limited number of spheroids Cells need to be pre-treated with magnetic beads. Beads are expensive. Beads at high concentration might be toxic for cells.