This research presents a novel Testis-on-a Chip- platform. Testicular cells are enzymatically isolated from the seminiferous tubules of sexually immature mice, seeded in a methylcellulose gel and cultured in a microfluidic chip. The unique design sandwiches the soft methylcellulose between stiffer agar support gels. The cells develop into organoids continuing to proliferate and differentiate. After seven weeks of culture the cells have over 95% viability. Confocal microscopy of the developed organoids reveals a structure containing the various stages of spermatogenesis up to and including meiosis II: premeiotic, meiotic and post-meiotic germ cells. The organoid structure also contains the supporting Sertoli and peritubular cells. The responsiveness of the system to the addition of testosterone and retinoic acid to the culture medium during the experiment are also investigated. As a benchmark, the Testis-on-a-Chip is compared to a conventional three-dimensional methylcellulose cell culture in a well plate. Analysis via FACS (Fluorescence-activated cell sorting) shows more haploid cells in the chip as compared to the plates. Immunofluorescence staining after seven weeks of culture shows more differentiated cells in the chip as compared to the well plate. This demonstrates the feasibility of our platform as well as its advantages. This research opens new horizons for the study and realization of spermatogenesis in-vitro. It can also enable the implementation of microfluidic technologies in future therapeutic strategies for pre pubertal male fertility preservation and adults with maturation arrest. Lastly, it can serve as a platform for drug and toxin testing.

Often, in semen samples with minute amounts of sperm, even the single spermatozoon required to fertilize an oocyte cannot be found in the ejaculate. This is primarily because currently, sperm is generally searched for manually under a microscope. In this study, dielectrophoresis (DEP) was investigated as an alternative automated technique for sorting sperm cells. Using a quadrupolar electrode array it was shown that the head and tail of the sperm had independent and unique crossover frequencies corresponding to the transition of the DEP force from repulsive (negative) to attractive (positive). These surprising results were further analyzed, showing that the head and tail have their own distinct electrical properties. This significant result allows for the sperm's head, which contains the DNA, to be distanced from potentially damaging high electric fields using negative DEP while simultaneously manipulating and sorting the sperm using the positive DEP response of the tail. A proof of concept sorting chip was designed and tested. The low crossover frequency of the tail also allows for the use of a higher conductivity, and thus more physiological, medium than the conventional DEP solutions. Although more research is required to design and optimize an efficient, user‐friendly, and high‐throughput device, this research is a proof of concept that DEP has the potential to automate and improve the processing of semen samples, especially those containing only rare spermatozoa.

Organ/organoid-on-a-chip (OoC) technologies aim to replicate aspects of the in vivo environment in vitro, at the scale of microns. Mimicking the spatial in vivo structure is important and can provide a deeper understanding of the cell–cell interactions and the mechanisms that lead to normal/abnormal function of a given organ. It is also important for disease models and drug/toxin testing. Incorporating active fluid flow in chip models enables many more possibilities. Active flow can provide physical cues, improve intercellular communication, and allow for the dynamic control of the environment, by enabling the efficient introduction of biological factors, drugs, or toxins. All of this is in addition to the fundamental role of flow in supplying nutrition and removing waste metabolites. This review presents an overview of the different types of fluid flow and how they are incorporated in various OoC models. The review then describes various methods and techniques of incorporating perfusion networks into OoC models, including self-assembly, bioprinting techniques, and utilizing sacrificial gels. The second part of the review focuses on the replication of spermatogenesis in vitro; the complex process whereby spermatogonial stem cells differentiate into mature sperm. A general overview is given of the various approaches that have been used. The few studies that incorporated microfluidics or vasculature are also described. Finally, a future perspective is given on elements from perfusion-based models that are currently used in models of other organs and can be applied to the field of in vitro spermatogenesis. For example, adopting tubular blood vessel models to mimic the morphology of the seminiferous tubules and incorporating vasculature in testis-on-a-chip models. Improving these models would improve our understanding of the process of spermatogenesis. It may also potentially provide novel therapeutic strategies for pre-pubertal cancer patients who need aggressive chemotherapy that can render them sterile as well asfor a subset of non-obstructive azoospermic patients with maturation arrest, whose testes do not produce sperm but still contain some of the progenitor cells.

DOI: https://doi.org/10.1002/elps.201800437 The cover picture shows a heterogeneous mixture of live sperm, dead sperm (stained with red fluorescent dye) and other cell types enter the microfluidic device. A focusing electrode guides them to the di‐electrophoresis‐based sorting electrode pair. The unwanted cells and debris are repelled by the electrode and leave via the upper channel while the live sperm`s tails (both motile and immotile) are pulled onto the electrodes guiding them to an alternate exit port. The inset shows the electric field intensity distribution. The fields are highest in the gap between the electrodes where the tail is trapped while the head on the other hand is distanced from the potentially harmful electric fields to enable safe sorting.

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