Table 2 |.

Parameters for spheroid formation

Parameters	Optimization	Examples	Steps
Seeding density	Spheroid density depends upon cell size, shape, morphology, and rate of proliferation. Various densities should be screened to achieve ~ 500 μm in spheroid diameter upon embedding into matrix. Importantly, the larger the cell number, the larger the spheroid, the greater oxygen differential between the external cells and the cells internal to the 3D structures.	3000 cells/well (H1299, 4T1) 1000 cells/well (A375)	Steps 16 – 18
Nanoparticle contamination	Sterile 96-well plates and/or sterile pipet tips are often contaminated with sterilized nanoparticles that can become embedded within a spheroid and deform its shape. 1.5X spheroids are created to account for unusable spheroids.		Steps 16 – 25
Cell adherence	Heterogeneous cells can express distinct adherence junction profiles to regulate cell – cell junctions and cell – matrix adhesion/interactions. Centrifugating 96-well plate places cells in the center of the well near one another to promote cell – cell junction formation. Upon spheroid formation, different matrices can be screened to determine the ability for cell – matrix adhesion formation and interactions.	rBM (H1299, A375) Collagen I (4T1)	Step 18
Time	After centrifugation, spheroid formation requires a 24 h or more incubation time. Cells can be screened to determine optimal incubation time to maintain both spheroid integrity during the embedding process and cell viability after.	72 h (H1299, 4T1, A375)	Step 18