Vortex-actuated pre-enrichment accelerates human regulatory T cell sorting and improves early fitness.

Cell selection is critical for regulatory T cell (Treg) therapy manufacturing. Pre-enrichment can reduce sorting time, yet data comparing different methods remain scarce. Highway1, a Good Manufacturing Practice (GMP)-compliant cell sorter, separates cells using transient vortices (i.e., vortex-actuated cell sorting [VACS]), which avoids exposure to magnetic nanoparticles and may improve cell viability compared to immunomagnetic separation (IMS). We compared VACS with IMS enrichment before sorting for human Tregs. Tregs were sorted from peripheral blood mononuclear cells isolated from donor leukocyte cones directly using the Highway1 (DP), CD4+CD25+ VACS enrichment then VACS (VP), or CD25+ IMS positive selection then VACS (25MP), and expanded with anti-CD3+/CD28+ stimulation beads, IL-2, and rapamycin. VP, 25MP, and DP produced Tregs of comparable purity. VP reduced sorting time but decreased yield compared to DP. VP resulted in fewer late apoptotic and hypoproliferative, IL-2 hyporesponsive cells, and generated Tregs that expanded more than 25MP. Phenotypic markers and suppression function did not differ after 21 days of expansion. CD4+CD25+ VACS enrichment reduced the time needed for Treg sorting compared to direct VACS and generated Tregs that expanded more than those enriched by CD25+ IMS selection, supporting adoption of a single-platform, closed VACS workflow and justifying validation at GMP scale.