Advancing urine‐derived stem cells: Cryopreservation validation and sex‐specific metabolism

BACKGROUND: Urine-derived stem cells (UDSC) are an emerging, non-invasive source of human stem cells combining easy collection, broad accessibility and high patient compliance with multilineage differentiation capacity. However, key gaps remain in UDSC research, particularly in understanding sex-related differences and the lack of a validated cryopreservation protocol, a critical aspect for primary cells, given their variability in colony formation, proliferation rates and experimental timing. To address these limitations, this study aimed to establish, for the first time, a reliable protocol for UDSC cryopreservation and to explore potential sex-related differences, with a specific focus on glycolysis and mitochondrial respiration. METHODS: UDSC were isolated from urine samples of healthy donors (aged 27-50, 4 males and 4 females), cultured in 1:1 DMEM:KSFM supplemented with 10% fetal bovine serum and cryopreserved at passages 2-4 using the same medium with the sole addition of 5% dimethyl sulfoxide. Cells were evaluated for viability, apoptosis/necrosis, metabolic profile and multilineage differentiation potential. Comparisons were performed based on donor sex, as well as before and after cryopreservation. RESULTS: Male- and female-derived UDSC displayed no significant differences in viability and cell death or metabolic profile. Moreover, supervised and unsupervised machine learning methods were unable to discriminate between the two groups, allowing for pooled data analysis and improved statistical power. Similarly, fresh and cryopreserved UDSC displayed comparable viability, metabolic activity and multilineage differentiation relative to fresh cells, with no detectable differences in computational analyses. CONCLUSIONS: These findings support UDSC adoption for biobanking, disease modelling and regenerative medicine.