Swift Formation Of Optimal Single Spheroids Towards In-Vitro 3-Dimensional Tumour Models

Introduction: Monolayer cell cultures, while useful for basic in vitro studies, are not physiologically relevant. Spheroids, on the other hand provide a more complex 3-dimensional (3D) structure which more closely resemble in vivo tumour growth thereby allowing results obtained with spheroids relating to proliferation, cell death, differentiation, metabolism, and various anti-tumour therapies to be more predictive of in vivo outcomes. Methods: The protocol herein presents a rapid and high throughput method for the generation of single spheroids whose applicability was demonstrated on various cancer cell lines including (U87 MG; SEBTA-027; SF188) brain cancer cells, (DU-145, TRAMP-C1) prostate cancer cells, and (BT-549, Py230) breast cancer in green coded 96-round bottom well plates. Results: Homogeneous compact spheroid morphology was evidenced as early as 24 hours after following the protocol. By using confocal microscopy and IncuCyte live imaging, the proliferating cells were traced in the rim and the dead cells were found inside the core region of the spheroid. H&E staining of spheroid slices and Western blotting were utilised to investigate the tightness of the cell packaging by adhesion proteins. Carnosine was used as an example of treatment for U87 single spheroids. Conclusions: This 5 step-protocol allows the rapid generation of spheroids, which will help towards reducing the number of tests performed on animals and encourage 3D modelling experiments from early-stage research.