Prediction of Mesenchymal Stromal Cell Immune Suppression Using Live Imaging in a Three-Dimensional Microfluidic...

Mesenchymal stromal cells (MSCs) hold significant promise for regenerative medicine and cell-based therapies due to their multipotency and immunomodulatory properties. A critical barrier to clinical translation is that traditional labor-intensive 2D endpoint assays have had limited success in predicting functional outcomes; in part because they fail to replicate the dynamic cell behaviors MSCs exhibit within complex 3D tissue microenvironments. Because MSC activity is closely tied to local biochemical and biophysical cues, we hypothesized that live imaging of MSCs in a biomimetic 3D environment could reveal functional phenotypes indicative of immunomodulatory potency. Here, we employed a high-throughput 3D microfluidic device coupled with real-time imaging to quantify morphological features and migration dynamics of MSCs from five donors and compared these to donor-matched immunosuppressive potency measured via a standard T-cell suppression assay. MSCs exhibited donor-specific differences in morphological features, including compactness, eccentricity, solidity, and extent. These variations were accompanied by distinct differences in their migratory behaviors as well. Notably, key 3D phenotypic features correlated strongly with immunomodulatory function. Overall, donors with higher T-cell suppression capacity displayed distinct morphological features, higher average migration, and higher levels of locomotion. Together, these results demonstrate that tissue-mimetic 3D microenvironments can elicit dynamic MSC phenotypes that are predictive of immunomodulatory function, highlighting the value of live-imaged microphysiological tissue models for linking in vitro cell behavior to functional therapeutic outcomes.