Cellular iron flux measurement by micromagnetic resonance relaxometry as a critical quality attribute of mesenchymal stromal cells.

BACKGROUND: Clinical application of mesenchymal stromal cells (MSCs) for cartilage regeneration has been hampered by their poor retention of chondrogenic potential during in vitro culture, which has led to highly variable cartilage repair outcomes. Consequently, there is an urgent need for a reliable assay to predict MSC chondrogenic potential during cell manufacturing. METHODS: In this study, we developed a nondestructive MSCs iron flux monitoring methodology using spent culture media, utilizing micromagnetic resonance relaxometry (µMRR). We demonstrated that the dynamics of iron uptake and release by MSCs in culture can be reliably inferred from iron concentration changes in culture medium, with an unprecedented temporal resolution (<1 hour). RESULTS: Analysis of 3 MSC donors across 6 independent culture batches revealed a significant correlation between iron homeostasis (iron flux during culture) and chondrogenic differentiation outcomes, while a departure from iron homeostasis (significant iron uptake and accumulation) was correlated with impaired chondrogenesis. By contrast, cell proliferation, although essential for manufacturing to achieve sufficient cell numbers, did not reliably correlate with chondrogenic capacity. Furthermore, ascorbic acid supplementation during culture, which is known to promote MSCs proliferation and chondrogenic quality, regulated iron homeostasis by limiting iron flux. CONCLUSION: Our findings identify iron homeostasis as a potential chondrogenic-associated critical quality attribute of MSCs. This rapid, nondestructive monitoring strategy offers a promising approach to improve manufacturing efficiency and consistency by providing real-time insight into cellular iron flux. In addition, the methodology here impacts broader iron biology by providing a time-resolved iron flux measurement that is not currently available.