Separation of Human P63⁺ Lung Progenitor Cells from Growth-Arrested Feeders Using Multidimensional Double Spiral...

Recent clinical trials have highlighted the potential of P63⁺ lung progenitor cell (LPC) transplantation for lung repair and regeneration. Currently, ex vivo P63⁺ LPC expansion depends on coculture with growth-arrested fibroblast feeder cells (GAFs), necessitating repeated purification during passaging. While differential enzymatic digestion (DED) and fluorescence- or magnetic-activated cell sorting techniques (FACS/MACS) offer partial solutions, a scalable, efficient, and consistent separation technique remains unmet, particularly for cell therapy manufacturing. Here, we present a multidimensional double spiral (MDDS) inertial microfluidic device designed for high-throughput label-free enrichment of P63⁺ LPCs. The MDDS device achieves cell size-based separation of P63⁺ LPCs and growth-arrested feeder cells, processing at a rate of 10⁶ to 10⁷ cells per minute. MDDS-sorted P63⁺ LPC purity correlates with the LPC-to-GAF ratio in culture. With an initial ratio >1:1, it yields P63⁺ LPC purity exceeding 80%. Moreover, the device consistently recovers >80% of P63⁺ LPCs, with the unrecovered fraction enriched in senescent cells exhibiting compromised clonogenicity and differentiation capacity. In direct benchmarking against DED and FACS, the MDDS device delivered a balanced performance in terms of purity and recovery, while offering advantages in throughput, consistency, and scalability. We propose that this technology could enable more consistent and efficient enrichment of feeder-cultured P63⁺ LPCs, thereby supporting more robust clinical manufacturing processes.