3D tumor cell cultures are currently emerging as the novel standard for cytotoxicity testing as well as in vitro molecular studies, but various solutions are available to generate them. The aim of this study was to test whether there is one-fits-all solution to generate tumor spheroids for further studies.
We used three breast tumor cell lines (MCF-7, MDA-MB-231, MDA-MB-361) and one glioblastoma cell line (U87), as comparison for MDA-MB-361 which is a secondary (metastatic) brain tumor. Different hydrogels (Matrigel® Corning, TrueGel3D -1, -6,-7 with or w/o RGD adhesion peptide® Merck, and TrueGel3D® HTS Hydrogel Plates®Merck) were tested for spheroid formation, as well as ultra-low attachment surface 24 well-plates ®Corning (no gels). Cells were seeded in two concentrations (300 and 3000/cm2) and documented daily for the first 5 days, than weekly for spheroid formation. Viability was tested using dual fluorescein diacetate/propidium iodide stain.
For higher cell concentrations, presence of spheroids can be documented as early as 5 days, but at least 7 days are recommended. Once formed, spheroids can be maintained more than 28 days, with twice a week cell medium change. Presence of organic molecules (basement membrane matrix or RGD peptide) in the gel impaired formation of tumor spheroids for aggressive cell lines (MDA-MB-261 and U87) and yielded mixed cultures (2D and spheroids) for the rest. Also, higher stiffness and the presence of a non-degradable cell linker prevented proliferation of cells and formation of spheroids, regardless of cell type. A crosslinker gradient, although favored formation of all tested breast cancer spheroids, interfered with formation of spheroids for U87 cells. Finally, ultra-low adherence plates allowed consistent formation of spheroids only for MCF-7 cells, whereas for the rest, irregular cell aggregates were observed at higher cell concentrations.
Dextran-based polymers can be used for tumor spheroid formation regardless of cell type, provided that no adhesion peptides are added.
Victor Babes National Institute of Pathology.
Ministry of Research, Innovation and Digitization, project no. COP A 1.2.3., ID: P_40_197/2016, PN 19.29.01.04 and 31PFE/2021, and CCCDI - UEFISCDI, project number PN-III-P2-2.1-PED-2019-3141, ctr. 382.
All authors have declared no conflicts of interest.