I need to know about the basic protocols that should be followed to do spheroidal assay as a model for cancer study?.what are the best cancer cell lines that can be used for the assay ?
Hey Zubin, recently we worked with spheroid model of 3D tumors. For protocols, you may follow our recent paper (Phys. Chem. Chem. Phys., 2016, 18, 18381). For further details, you may go through reference 23 of our paper.
In our paper, we worked with HeLa and A549 spheroids. I think HeLa is a good option.
Hope this helps.
Cheers
A selection of invasive cell lines with a high proliferation capacity would be a good start. MDA-MB-231 and 4T1 breast cancer cell lines are good at forming spheroids.
find the attached protocol. i follow the same for other spheroid cultures.
Hello,
several possibilities exist for preparing spheroids: low-adherent plates, commercial plates or coated with poly-HEMA (https://www.ncbi.nlm.nih.gov/pubmed/22043235), hanging drops (InSphero for ex.), embedding in a matrix (QGel matrix from www.qgelbio.com, Matrigel coated plates...).
For the cell line, it depends on the cancer you want to study. Some cells will not form spheroids in certain conditions (LN-229, glioblastoma cells, do not form spheroids, neither in low-adherent plates nor in hanging drops, but PANC-1, pancreatic cancer cells, work well). Co-culture with another cell line, such as fibroblasts, can be very beneficial for the tumoral cells and to get closer to the in vivo tumor (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127948).
For viability testing, CellTiterGlo-3D from Promega is an easy-to-use, luminescent assay (https://www.promega.com/-/media/files/resources/protocols/technical-manuals/101/celltiter-glo-3d-cell-viability-assay-protocol.pdf) but cells are sacrificed for performing the assay. For long term culture, you can use almarBlue viability test, as it is not cytotoxic and cells growth can be continued after washing the dye (10% alamarBlue in medium, 3h incubation at 37°C+5%CO2, fluorescence reading compared with a blank well with no cells. Wash 3x15min with medium and incubate cells for further growth).
Confocal microscopy is also a good tool to compare conditions and visualize the cells. Cells can be fixed with 0.4% PFA.
Hope this helps.
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