Glioblastoma multiforme (GBM) is the most malignant primary form of glioma with prognosis of around a year after diagnosis ( Jiang and Uhrbom, 2012 Ostrom et al., 2018).

The protocol allows the rapid generation of spheroids, which will help towards reducing the number of tests performed on animals. Carnosine was used as an example of treatment for U87 single spheroids. The H&E stain of spheroid slices and the western blotting were utilised to investigate the tightness of the cell packaging by adhesion proteins. By using confocal microscopy, the proliferating cells were traced in the rim and the dead cells were found inside the core region of the spheroid. Cells are washed with anti-adherent solution, and the homogeneous compact spheroid morphology was evidenced as early as 24 hours after 10 minutes centrifugation for the seeded cells. The protocol herein presents a novel and rapid generation for single spheroids of various cancer cell lines, U87 MG SEBTA-027 SF188, brain cancer cells, DU-145, TRAMP-C1, prostate cancer cells, in 96-round bottom well plates. However, the cost associated with their generation often involve expensive, plate, media, and growth supplements, which have limited their use for high throughput experiments. Spheroids, on the other hand provide a more complex 3-dimensional (3D) structure which more resemble the in vivo tumour growth thereby allowing results obtained with those on proliferation, cell death, differentiation, metabolism, and various anti-tumour therapies to be more predictive of in vivo outcomes. Monolayer cell culture, while useful for basic in vitro studies, are not physiologically relevant.

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