Viscoelastic Focusing and Sorting of Particles and Asymmetric Cells in Straight Microfluidic Channels

Worldwide, infertility affects more than 70 million couple1–3 and it has been reported that 50% of these cases4 can be accounted for male patients; among these men, 10 to 15% suffer from azoospermia4, which is defined as the lack of sperm in the ejaculate sample5. Patients suffering from nonobstructive azoospermia (NOA) can still have children thanks to procedures such as testicular sperm extraction (TESE) from a testicular biopsy sample6,7 but the presence of an abundance of red blood cells (RBCs) remain the main impediment in this technique8. Indeed, the isolation of the few spermatozoa from the sample has to be performed manually as filtration methods are inefficient and also time consuming6. Currently, there is no device that is able to perform erythrocytes-sperm separation in an efficient, cost-effective, and clinically feasible way. Here, we demonstrate a simple microfluidic straight channel with a rectangular cross-section, which is able to separate sperm cells from erythrocytes by exploiting the viscoelastic effect of the medium. Our device was made in polydimethylsiloxane with the classic soft-lithography procedures, making it simple and low cost. We extensively investigated multiple channels with different geometrical characteristics (100 μm x 50 μm, 50 μm x 25 μm, 25 μm x 50 μm, 75 μm x 25 μm) using spherical polystyrene particles of different sizes (15, 10, 7, 4, 2 μm diameter) as cell surrogates. This was done to find the optimal geometrical and flow conditions in which the asymmetric cell separation could be possible. During our investigation, we optimized the concentration of the viscoelastic fluid needed for the focusing, the channel length at which both particles and cells showed stable equilibrium positions, and the flow conditions for the optimum performance. We also investigated the 3D behavior of particles inside 50 μm x 25 μm channel and we were able to test RBCs and sperm cells together in a 75 μm x 25 μm channel, demonstrating a high efficiency of separation (> 86% for RBCs and > 59% for spermatozoa). Finally, in the open outlets design channel, we demonstrated the perfect (~ 100%) purity and efficiency of sorting with 4 and 7 μm particles at flow rates lower than 5 μL/min. Ultimately, our study demonstrates high purity separation and collection of spermatozoa is possible, making our device suitable for the clinical application of in vitro fertilization procedures.