Differential Phase Contrast Imaging to Predict MSC Immune Function

Abstract Mesenchymal stromal cells (MSCs) exhibit significant immunomodulatory potential, making them promising candidates for cell‐based therapies in autoimmune and inflammatory diseases. However, the heterogeneity of MSC cultures and a lack of robust, predictive potency assays have hindered their clinical translation. In this study, the potential of single‐cell morphological imaging during MSC expansion is explored as a method to estimate indoleamine‐2,3‐dioxygenase (IDO) protein and enzyme activity, a common immunosuppressive capacity measure. Fluorescence and label‐free quantitative differential phase contrast (qDPC) imaging is employed to non‐invasively extract morphological features from live MSCs during biomanufacturing with machine learning (ML) regression models to predict single cell IDO activity. qDPC imaging characterization is extended to estimate a previously established consensus model of MSC potency based on their IDO activity and immune suppression on T cells. These findings establish a foundation for scalable, non‐destructive monitoring of MSC immunomodulatory capacity, facilitating the future development of quality control strategies for MSC manufacturing and clinical applications.