Table 2.

State of the art of methods for the generation and culture of cancer spheroids and organoids: advantages and inconvenient.

Method or Technology	Advantages	Inconvenient
Hanging drop method	Simple and easy to perform Low cost Generate small numbers of organoids No need for specialized equipment	Can be technically challenging to handle and manipulate the organoids Limited scalability and reproducibility Takes a long time Difficult long-term culture
Low-attachment plate method	Can be used to generate small or large numbers of organoids. Simple and easy to perform Wide range of cell types and seeding concentrations	Formation of spheroids with irregular and heterogeneous shapes and sizes Not provide adequate oxygen and nutrient supply to the center of the spheroids Cost of the equipment Continuous constant agitation could be a need to culture spheroids and organoids Challenging medium exchanges
Microwell method	Allows for the formation of multiple organoids in a single well Allows for high-throughput screening Allows for better control over the microenvironment of the organoids Allows for the formation of multiple organoids in a single well	Can be technically challenging to handle and manipulate the organoids High cost of the equipment Can be technically challenging to handle and manipulate the organoids Relatively high cost of the equipment
Magnetic levitation	Formation of uniform cell aggregates Can be used to create dynamic and versatile microenvironments for cells, (ex: mimicking the mechanical forces of blood flow) Using magnetic fields to manipulate the position of cells in real time	New technology and more research are needed to fully understand its potential uses in cancer research High cost of the specialized equipment and the magnetic beads Difficult to scale up to larger numbers of cells or larger cell cultures Cell type compatibility of magnetic beads May require additional steps to remove the beads after the cells have formed spheroids or organoids
Rotatory systems (Bioreactors and spinner flask)	Can generate organoids in a controlled and reproducible manner Mimic the in vivo microenvironment of tumors Can culture large numbers of organoids in a small volume of medium	Complexity of the technology High cost of the equipment Need of very specialized equipment Can be difficult to handle and manipulate the organoids High shear stress and for cells
Extracellular-matrix-based method	Can mimic the in vivo microenvironment of tumors Mimicking cell-to-ECM interactions Improvement of the TME supportive and physiologically relevant environment for the cells to grow and differentiate	Can be technically challenging to handle and manipulate the organoids High cost of ECM products Extensive batch-to-batch variability Poor control of mechanical properties Choice of the appropriate ECM