Sperm recovery from testicular biopsies containing tissue fragments and other somatic cells, using an inertial spiral microfluidic chip.

Severe male-factor infertility, particularly non-obstructive azoospermia, often requires recovery of rare sperm from testicular biopsy samples that contain large tissue fragments and dense background cells. Conventional microscopic identification is labor-intensive, time-consuming, and prone to human error, while existing microfluidic approaches are susceptible to channel blockage by tissue debris. Here, we present a two-stage passive microfluidic platform for rapid, label-free sperm isolation from biopsy-derived cell suspensions. The system combines a 40 μm pre-filtration stage to remove large tissue fragments with an inertial spiral microfluidic separator optimized through finite element simulations while operating at Reynolds numbers of 10-15. The device exploits the balance between inertial lift and Dean drag forces to achieve size-based cell focusing and separation without external fields. Experimental tests using both handmade cell suspensions and clinical fine-needle aspiration (FNA) samples demonstrated good sperm recovery efficiencies exceeding 90%, while effectively removing more than 90% of red blood cells, other somatic cells, and all large tissue fragments at the optimal flow rate. The separation process preserved sperm motility, morphology, and DNA integrity, even after repeated processing. Notably, sperm with higher motility, more normal morphology, and lower DNA fragmentation index were preferentially separated, indicating that the system not only enables efficient cell separation but also inherently enables the selection of higher-quality sperm. Application to FNA samples reduced sperm retrieval time from several hours to less than 13 minutes and consistently enriched morphologically normal sperm at the target outlet. This two-stage passive microfluidic separator offers a robust and clinically relevant solution for the selection of sperm from complex biopsy samples and represents a significant step toward automated sperm preparation for assisted reproductive technologies.