Passage-Dependent Size Distributions of Human-Derived Cells: Implications for Metabolic Assays.

Biological noise is ubiquitous in living systems; yet, it is often neglected in cell-based experiments, potentially biasing data interpretation. We provide a quantitative characterization of single-cell equivalent diameter distributions in six human cell types using cell counting. By analyzing thousands of cells over passage number, we show that cell size is phenotype dependent and varies significantly with passage, with most cell types exhibiting a progressive reduction in median diameter. This variability is structured: When diameters are converted to masses, the distributions obey scaling laws, revealing conserved statistical properties of human cells in culture. Because key physiological processes such as metabolism scale with cell mass, passage-dependent shifts in diameter distributions can propagate into functional readouts. We show that changes in cell size are sufficient to bias estimates of construct-level metabolic rate, potentially confounding the interpretation of size-normalized assays and treatment effects. Our results highlight that biological noise in vitro is a source of statistical structure that enables scaling analyses and a dynamic property that, if ignored, can lead to systematic misinterpretation of experimental outcomes. Accounting for size distributions and their evolution over passages may therefore improve experimental design, data interpretation, and, ultimately, the translatability of in vitro models.