P-015 Automated, efficient sorting of sperm using dielectrophoresis (DEP), utilizing the electrical properties of the tail as a biomarker of the viability, even for immotile cells

Abstract

Abstract Study question Developing a simple and automated method and device that sorts semen cell by cell for eventual application to retrieving rare sperm from severely oligozoospermic samples. Summary answer Over 90% of the viable cells in a sample can be sorted out from non-viable sperm cells and other debris using this method. What is known already The varying electrical properties of different cell types can result in unique frequency dependent dielectrophoretic (DEP) behavior when they are exposed to alternating current (AC). They are either repelled or attracted depending on their properties and the field frequency. This has been applied to sorting rare cells such as circulating tumor cells from blood. We have previously shown that the head and tail of human sperm have different electrical properties. As a result, at certain frequencies of an AC electric field the tails of viable cells (even immotile ones) are attracted to electric field gradients while the heads are distanced. Study design, size, duration Semen samples from several patients at Rambam medical center were collected and cryopreserved. Later the thawed semen samples were then tested in our sorting device. At least one hundred live cells were evaluated in each sample. The percentage of live cells that were successfully sorted out of the original mixture of live sperm cells, dead sperm cells and other debris was assessed. Participants/materials, setting, methods After thawing the samples were stained using CFDA and PI for visual live/dead identification (in actual practice no staining is necessary). The device consists of a polydimethylsiloxane (PDMS) microchannel on a glass slide patterned with Indium Tin Oxide (ITO) transparent electrodes. In the device the sperm are transferred to a low conductivity buffer. A curved electrode then sorts them out of the stream of the cell mixture to an alternate exit. Main results and the role of chance We have successfully demonstrated a new biomarker of live sperm (even immotile ones). The tails of live sperm have a positive (attractive) DEP response while dead sperm react negatively and are repelled from field gradients. At the specific frequencies used the head is simultaneously distanced from the electric fields helping to prevent damage to the DNA during the sorting. We also showed that this effect could be used to sort sperm with over 90% efficiency from a sample with dead sperm and other debris. Since each cell passes the electrodes and is automatically sorted it could possibly be used for severely oligozoospermic and cryptozoospermic samples to find sperm and make more sperm available for more discriminatory sperm selection in such samples. Nowadays such samples are searched manually and the minute number of sperm present is often missed leading to a mistaken azoospermic diagnosis and possibly unnecessary surgery. The components are relatively low cost and the chips disposable allowing for wider implementation. Significant progress was also made in preventing sperm from sticking to the substrates using coatings and a vibrational motor. Additionally, a proof of concept parallelization of the electrodes to increase the throughput was also demonstrated. Limitations, reasons for caution The inherent difficulty with microfluidic technologies is low throughput. Further work is needed to effectively parallelize and optimize the system. Effective pre-processing to clean the sample may also help to increase the throughput. Wider implications of the findings This biomarker can potentially be useful in determining an immotile cell’s viability for ICSI. This technology can also be of potential use for testicular tissue samples where there are many other cell types and debris and it is difficult to find rare sperm. Advanced sperm selection can also be incorporated. Trial registration number not applicable