A microfluidic approach to rapid sperm recovery from heterogeneous cell suspensions

Abstract

The isolation of sperm cells from background cell populations and debris is an essential step in all assisted reproductive technologies. Conventional techniques for sperm recovery from testicular sperm extractions stagnate at the sample processing stage, where it can take several hours to identify viable sperm from a background of collateral cells such as white bloods cells (WBCs), red blood cells (RBCs), epithelial cells (ECs) and in some cases cancer cells. Manual identification of sperm from contaminating cells and debris is a tedious and time-consuming operation that can be suitably addressed through inertial microfluidics. Microfluidics has proven an effective technology for high-quality sperm selection based on motility. However, motility-based selection methods cannot cater for viable, non-motile sperm often present in testicular or epididymal sperm extractions and aspirations. This study demonstrates the use of a 3D printed inertial microfluidic device for the separation of sperm cells from a mixed suspension of WBCs, RBCs, ECs, and leukemic cancer cells. This technology presents a 36-fold time improvement for the recovery of sperm cells (> 96%) by separating sperm, RBCS, WBCs, ECs and cancer cells into tight bands in less than 5 min. Furthermore, microfluidic processing of sperm has no impact on sperm parameters; vitality, motility, morphology, or DNA fragmentation of sperm. Applying inertial microfluidics for non-motile sperm recovery can greatly improve the current processing procedure of testicular sperm extractions, simplifying the fertility outcomes for severe forms of male infertility that warrant the surgery.

Microfluidic Systems for Isolation of Spermatozoa from Testicular Specimens of Non-Obstructive Azoospermic Men: Does/Can It Improve Sperm Yield?

“…The authors were able to demonstrate separation and isolation of spermatozoa from WBCs. More recently, Vasilescu and coworkers [49] used a similar spiral microchannel device produced by 3D printing to demonstrate rapid spermatozoa recovery from heterogeneous cell suspension of spermatozoa, WBCs, red blood cells, epithelial cells, and leukemic cancer cells. This study demonstrated rapid (5 min) separation of spermatozoa from other cell types and, very importantly, that this spiral microfluidic processing had no detrimental influence on spermatozoa viability, morphology, or DNA integrity.…”

“…Integration of multiple technologies-existing and of the future-will likely facilitate the use of microfluidics for improving success, reducing technical signatures and variation, and providing bridges over current limitations. Potential examples include combined spiral microfluidics [48,49] with subsequent short-term culture to assess viability/motility [20] and Raman spectroscopy to non-invasively interrogate sperm DNA integrity [52,53].…”

Multi-dimensional-double-spiral (MDDS) inertial microfluidic platform for sperm isolation directly from the raw semen sample

“…Many of these microfluidic technologies have the drawback of low throughput for sample preparation, allowing just small quantities of sperm to be processed, in the order of a few μL to less than 500 μL, while the number of sperm capable of being processed depends on the dilution condition required for the operation of each microfluidic technology. To overcome this limitation, some inertial spiral microfluidic devices have recently been applied for sperm isolation and successfully demonstrated leukocyte removal with higher throughput and sperm recovery compared to the conventional methodologies 17 , 18 , 26 , 27 . In the inertial microfluidics requiring a high flow rate (order of mL/min) with a finite Reynolds number ( , where , , and represent the density, dynamic viscosity, and maximum velocity of the fluid, respectively, and represents the hydraulic diameter of the channel) 28 – 30 , particle behavior in the microchannel is dominantly affected by inertial lift forces.…”

“…Vasilescu et al . introduced a 3D printed inertial microfluidic device and successfully demonstrated the separation of sperm cells from blood cells, epithelial cells, and leukemic cancer cells with high sperm recovery (> 96%) 27 . Although the recent works successfully showed that the inertial microfluidic devices can handle a large volume of a seminal cell sample with relatively high sperm recovery, the separation efficiency is still insufficient and should be improved to increase output quality.…”

“…Seminal cells encompass just a small fraction of semen (~ 5% of volume in semen comes from the epididymis containing the sperm cells) 31 ; therefore, it is important to consider other seminal parameters. Washed seminal cells or washed semen samples have been used to determine device performance in all previous works involving inertial microfluidics 17 , 18 , 26 , 27 , which is the main drawback to limit the technology’s clinical impact because the pre-washing step requires manual and time-consuming centrifugation. A useful system for clinical applications should demonstrate high performance regardless of the unique properties of a given (semen) sample without any additional washing steps.…”