Abstract
From growing cells in spheroids to arranging them on complex engineered scaffolds, three-dimensional cell culture protocols are rapidly expanding and diversifying. While these systems may often improve the physiological relevance of cell culture models, they come with technical challenges, as many of the analytical methods used to characterize traditional two-dimensional (2D) cells must be modified or replaced to be effective. Here we review the advantages and limitations of quantification methods based either on biochemical measurements or microscopy imaging. We focus on the most basic of parameters that one may want to measure, the number of cells. Precise determination of this number is essential for many analytical techniques where measured quantities are only meaningful when normalized to the number of cells (e.g. cytochrome p450 enzyme activity). Thus, accurate measurement of cell number is often a prerequisite to allowing comparisons across different conditions (culturing conditions or drug and treatment screening) or between cells in different spatial states. We note that this issue is often neglected in the literature with little or no information given regarding how normalization was performed, we highlight the pitfalls and complications of quantification and call for more accurate reporting to improve reproducibility.