Optimization and Parallelization of Sorting by Interfacial Tension (SIFT) for High-Throughput Metabolic Cell Sorting.

A systematic optimization of throughput and operational run time in Sorting by Interfacial Tension (SIFT) is presented. Reducing droplet size and enabling a broader distribution of droplet trajectories increased the number of droplets processed per sorting element, resulting in about a four-fold improvement in throughput from 30 to 125 droplets per second. Throughput was further enhanced through device parallelization, as demonstrated by devices incorporating two and four independent sorting regions. These configurations distribute droplets evenly across sorting elements. The elements exhibited comparable pH sorting thresholds, indicating similar flow conditions and drag forces within each region. Among the designs evaluated, the two-element configuration provided the optimal balance of throughput, specificity, robustness, and simplicity. It achieved maximum throughputs of about 250 droplets per second. In many biological applications, only 1 in 20-30 droplets are occupied to minimize multiple-cell occupancy, resulting in an effective sorting rate of approximately 8 cells per second. Throughput and pH sorting thresholds were preserved for two hours of continuous cell sorting. The improved platform was applied to examine the relationship between cellular glycolysis and iron homeostasis at the single-cell level in activated Jurkat cells. It revealed a subpopulation of highly glycolytic cells with significantly elevated iron uptake, consistent with prior reports linking iron regulation and T cell metabolism. Collectively, these advances expand the scale, stability, and biological applicability of SIFT. These advances facilitate large-scale functional studies and the capture of rare, metabolically distinct, cell populations.