I have been generating spheroids from HNSCC cell lines. During spheroid formation, some of the cells may not form spheroids. So how can we seperate these single cells and collect only spheroids for further experiments? Can we do differential centrifugation? If yes, what is the speed required?
Depends on which method you use for growing the spheroids and also on what you are planning to do with the spheroids. We transfer spheroids from the 96-well plate where they have been cultured to sterile microcentrifuge tubes using a pipette and pipette tips with enlarged aperture. With a pair of scissors, enlarge the opening of the pipette tip by cutting approximately 2-3 mm off the tip. Before use, clean and disinfect the blades of the scissors with 80% ethanol. To successfully aspirate a spheroid, immerse the end of the pipette tip in the supernatant and move as close as possible to the spheroid without touching it, then gently but assertively retract the plunger to generate a fluid flow with which the spheroid will move into the tip. Gently release the spheroid into the tube avoiding to damage it. From: Spoerri L, Beaumont KA, Anfosso A, Haass NK* (in press): Real-time cell cycle imaging in a 3D cell culture model of melanoma. Methods Mol Biol, accepted for publication. [in Koledova (ed.) 3-D Cell Culture: Methods and Protocols, Springer Science+Business Mediam LLC, New York].
You can also check out these two (both on my RG account):
Smalley KS, Lioni M, Noma K, Haass NK, Herlyn M (2008; Epub 2007 Dec 19). In vitro three-dimensional tumor microenvironment models for anticancer drug discovery. Expert Opin Drug Discov 3: 1-10
Beaumont KA, Anfosso A, Ahmed F, Weninger W, Haass NK (2015) Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids. J Vis Exp 106: e53486, pp. 1-9, doi:10.3791/53486
Help with cancer cell spheroid formation and 3D invasion assay? - ResearchGate. Available from: https://www.researchgate.net/post/Help_with_cancer_cell_spheroid_formation_and_3D_invasion_assay#view=57dbf38f5b495276e55ee622 [accessed Jan 20, 2017].
Hello Gangotri!
Unlike the spheroids, single cells require high speed centrifugation to settle down in the tube bottom. Therefore if you use a suitable low speed for short time, the spheroids will settle down in the bottom. Wherease the single cells will remain in the supernatant that can be discarded accordingly.
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