Application of Inertial Microfluidics for Isolation and Removal of Round Spermatids from a Spermatogenic Cell Sample to Assist In-Vitro Human Spermatogenesis

<i>In-vitro</i> spermatogenesis holds great potential in addressing male infertility, yet one of the main challenges is separating round spermatids from other germ cells in spermatogonial stem cell cultures. STA-PUT, a method based on velocity sedimentation, has been extensively tested for this application. Though somewhat effective, it requires bulky, expensive equipment and significant time. In contrast, the method of inertial microfluidics offers a compact, cost-effective, and faster alternative. In this study, we designed, fabricated, and tested a microfluidic spiral channel for isolating round spermatids and purifying spermatogenic cells. A commercially available spiral device close to the calculated specifications was tested for rapid prototyping, achieving 79% purity for non-spermatid cells in a single pass, with ability to achieve higher purity through repeated passes. However, the commercial device's narrow outlets caused clogging, prompting the fabrication of a custom polydimethylsiloxane device matching the calculated specifications. This custom device demonstrated significant improvements, achieving 86% purity in a single pass compared to STA-PUT's 38%, and that without any clogging issues. Further purification could be attained by repeated passes, as shown in earlier studies. This work underscores the efficacy of inertial microfluidics for efficient, high-purity cell separation, with the potential to revolutionize workflows in <i>in-vitro</i> spermatogenesis research.