EEM fluorescence data-driven determination of cell density in adherent cell cultures

Accurate, precise, and non-invasive determination of cell density is a critical challenge in cell culture research, directly influencing experimental reproducibility and reliability of biological interpretations. Conventional methods, such as cell counting and colorimetric assays, are invasive and often unsuitable for continuous monitoring. This study evaluates excitation-emission matrix (EEM) fluorescence spectroscopy (multispectral fluorescence) combined with chemometric analysis as an innovative, data-driven approach for quantifying cell density in adherent cultures. The experiments were performed using two adherent cell lines, i.e., HaCaT and A375, simultaneously, to verify if the observed trends might extend beyond a single cell line and thus not be cell-line specific, which could further support the general applicability of the proposed cell number determination strategy. The number of cells in culture was determined using two reference methods, i.e., classical cell counting or the MTT assay, providing information on the number of metabolically active cells. Unfolded partial least squares (PLS) regression was used to develop models for predicting cell numbers based on information encoded in excitation-emission spectra of cell cultures. Statistical analysis shows that multispectral fluorescence surpasses cell counting in sensitivity, while the MTT assay exhibits the highest precision. By enabling label-free, non-destructive, and repeatable measurements with applicability across different cell lines, multispectral fluorescence can be proposed as an interesting alternative, especially for automated and dynamic monitoring of cell cultures.