Femtosecond label-free imaging empowers mesenchymal stem cells quality control

Mesenchymal stem cells (MSCs) hold significant promise in regenerative medicine, yet their clinical application is hindered by challenges such as cellular heterogeneity and quality control. This study aims to develop a rapid, non-invasive method for evaluating MSCs quality using femtosecond laser label-free imaging (FLI). We examined the proliferation, metabolic dynamics, and differentiation potential of MSCs from various tissue sources, including human dental pulp, umbilical cord, and fat, across different passages. Our findings indicate that with the passages increasing, the morphology of MSCs alters, proliferation capacity decreases, β-galactosidase activity linked to aging rises, and both osteogenic and adipogenic differentiation abilities markedly decline. FLI technology effectively captures these changes: reduced NAD(P)H/FAD ratio in the cells of higher passage suggests decreased metabolic activity, while enhanced aging-related fluorescence signals, such as lipofuscin, align with cellular senescence. During differentiation, increased fluorescence intensity of NAD(P)H and FAD signals heightened metabolic activity within the cells. Due to varying differentiation potentials among cells from different sources, NAD(P)H and FAD change patterns also differ. Difference in the optical REDOX ratio (FAD/(NAD(P)H+FAD)) among differentiation directions indicate that differentiation potential of MSCs correlates with metabolic reprogramming. Three-dimensional FLI of suspension cells further revealed that the cells of lower-passage exhibit greater spatial heterogeneity in metabolic signals, possibly reflecting more active mitochondrial function. This study confirms that FLI technology can effectively assess the proliferation activity, senescence, and differentiation potential of MSCs through non-invasive, dynamic monitoring of their metabolic status and morphological features, offering a novel approach for standardized quality assessment of MSCs preparations.