In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole.

Three-dimensional aggregation and organ culture methods are critical for recreating in vivo cellular phenomena outside the body. Previously, we used the conventional gas liquid interphase organ culture method to induce complete mouse spermatogenesis. After incorporating microfluidic systems, we achieved a significant increase in efficiency and duration of spermatogenesis. One of the major drawbacks preventing the popularization of microfluidics, however, is the use of a power-pump to generate medium flow. In this study, we produced a pumpless microfluidic device using hydrostatic pressure and a resistance circuit to facilitate slow, longer lasting medium flow. During three months of culture, results in induction and maintenance of spermatogenesis showed no difference between pumpless and pump-driven devices. Correspondingly, the spermatogonial population was favorably maintained in the pumpless device compared to the conventional method. These results show the advantage of using microfluidic systems for organ culture experiments. Our pumpless device could be applied to a variety of other tissues and organs, and may revolutionize organ culture methods as a whole.Spermatogenesis is a complex process of cell proliferation and differentiation, which is difficult to recapitulate ex vivo. In 2011, we cultured mouse testis tissues with the classical organ culture method that depends on the air-liquid interphase principle, and succeeded in the production of fertile sperm from spermatogonial stem cells 1,2 . However, the efficiency and duration of in vitro spermatogenesis were low and limited, respectively, being totally incomparable with those in vivo. In order to improve the culture system, we introduced microfluidic technology derived from a technique to manufacture semiconductors. In our microfluidic device, the culture medium flowed from the reservoir tank to the exit through a narrow channel. The testis tissue was spread in a shallow chamber of the device separated by a porous membrane from the flowing medium. Molecules of both nutritionals and waste-products diffused between the chamber and channel through the porous membrane to support the metabolism of the tissue. Oxygen could reach the tissue through the substrate of the device, polydimethylsiloxane (PDMS), which is highly oxygen-permeable. This microfluidic device maintained the two essential functions of the testis, spermatogenesis and testosterone production, for more than 6 months 3 . In addition, the device provided higher visibility of the tissue in culture, which even allows the identification of meiotic and haploid cells when GFP markers are incorporated. Thus, it enables us to examine the details of spermatogenic progression at cellular and even sub-cellular levels.The microfluidics system is promising for improving organ culture experiments, involving not only testis tissue but possibly other tissues and organs as well. However, there are several limitations which impede its popularization. One of them is the external syringe pump for the flow of medi...

In vitro spermatogenesis (IVS) using air–liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.

This study investigated the correlation between sperm motion parameters obtained by a computer-assisted semen analyzer and levels of reactive oxygen species in unwashed semen. In total, 847 patients, except for azoospermic patients were investigated. At the time of each patient's first consultation, semen parameters were measured using SMAS™ or CellSoft 3000™, and production of reactive oxygen species was measured using a computer-driven LKB Wallac Luminometer 1251 Analyzer. The patients were divided into two groups: reactive oxygen species - positive and negative. The semen parameters within each group were measured using one of the two computer-assisted semen analyzer systems and then compared. Correlations between reactive oxygen species levels and sperm motion parameters in semen from the reactive oxygen species - positive group were also investigated. Reactive oxygen species were detected in semen samples of 282 cases (33.3%). Sperm concentration (P < 0.01; P < 0.01), motility (P < 0.01; P < 0.05), and progressive motility (P < 0.01; P < 0.01) were markedly lower in the reactive oxygen species - positive group than in the reactive oxygen species - negative group. Among the sperm motion parameters in the reactive oxygen species - positive group, sperm concentration (P < 0.01; P < 0.01), motility (P < 0.05; P < 0.01), mALH (P < 0.05; P < 0.01), and progressive motility (P < 0.05; P < 0.01) also showed inverse correlations with the logarithmic transformed reactive oxygen species levels. Therefore, this study demonstrated that excessive reactive oxygen species in semen damage sperm concentration, motility, and other sperm motion parameters.

Purpose Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients. Methods Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of Acr‐Gfp, which reflects the progression of spermatogenesis. Results Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids. Conclusions The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.