Reproducibility warning: The curious case of polyethylene glycol 6000 and spheroid cell culture.

Reproducibility of results is essential for a well-designed and conducted experiment. Several reasons may originate failure in reproducing data, such as selective reporting, low statistical power, or poor analysis. In this study, we used PEG6000 samples from different distributors and tested their capability inducing spheroid formation upon surface coating. MALDI-MS, NMR, FTIR, and Triple SEC analysis of the different PEG60000s showed nearly identical physicochemical properties different, with only minor differences in mass and hydrodynamic radius, and AFM analysis showed no significant differences in the surface coatings obtained with the available PEG6000s. Despite these similarities, just one showed a highly reproducible formation of spheroids with different cell lines, such as HT-29, HeLa, Caco2, and PANC-1. Using the peculiar PEG6000 sample and a reference PEG6000 chosen amongst the others as control, we tested the effect of the cell/PEG interaction by incubating cells in the PEG solution prior to cell plating. These experiments indicate that the spheroid formation is due to direct interaction of the polymer with the cells rather than by interaction of cells with the coated surfaces. The experiments point out that for biological entities, such as cells or tissues, even very small differences in impurities or minimal variations in the starting product can have a very strong impact on the reproducibility of data.