A synergic approach to enhance long-term culture and manipulation of MiaPaCa-2 pancreatic cancer spheroids.

Tumour spheroids have the potential to be used as preclinical chemo-sensitivity assays. However, the production of three-dimensional (3D) tumour spheroids remains challenging as not all tumour cell lines form spheroids with regular morphologies and spheroid transfer often induces disaggregation. In the field of pancreatic cancer, the MiaPaCa-2 cell line is an interesting model for research but it is known for its difficulty to form stable spheroids; also, when formed, spheroids from this cell line are weak and arduous to manage and to harvest for further analyses such as multiple staining and imaging. In this work, we compared different methods (i.e. hanging drop, round-bottom wells and Matrigel embedding, each of them with or without methylcellulose in the media) to evaluate which one allowed to better overpass these limitations. Morphometric analysis indicated that hanging drop in presence of methylcellulose leaded to well-organized spheroids; interestingly, quantitative PCR (qPCR) analysis reflected the morphometric characterization, indicating that same spheroids expressed the highest values of CD44, VIMENTIN, TGF-β1 and Ki-67. In addition, we investigated the generation of MiaPaCa-2 spheroids when cultured on substrates of different hydrophobicity, in order to minimize the area in contact with the culture media and to further improve spheroid formation.