Isolation of adipose stromal cells from blood using a two-step microfluidic platform ASCfinder

Mesenchymal stromal cells (MSCs) hold significant promise for their therapeutic potential and their possible role as disease biomarkers. While evidence suggests the presence of circulating Adipose-derived MSC (ASC) in peripheral blood (PB), isolating them is particularly challenging due to their low abundance, size variability, and incomplete characterization of their native immunophenotype in PB. Consequently, the relationship between ASC frequency in blood and various physiological or pathological conditions has been underexplored. In this study, we introduce ASC-Finder, a label-free isolation method specifically designed for adipose stromal cells (ASCs), a key MSC population. ASC-Finder integrates two independent modules: a size-dependent hydrodynamic filtration unit for sorting erythrocytes directly from PB and a negative enrichment module based on immunological markers to deplete remaining leukocytes. The device enabled removal of 99.98% of erythrocytes while achieving high recovery rates of spiked ASCs (> 81%) at rare-event concentrations (< 100 ASC/mL blood). Remarkably, ASC-Finder operates without clogging, even after multiple runs with donor blood samples. Crucially, our method bypasses the need for harsh lysis, centrifugation, or dilution buffers, preserving both cell integrity and phenotype-key factors for the discovery of novel cellular events. This work represents a significant advancement in the direct enrichment of circulating ASCs from whole PB without cell lysis, offering a crucial step toward investigating the characterization and role of blood-circulating ASCs.