spermatogonial stem cells (ssCs) are the only stem cells in the body that transmit genetic information to the next generation. The long-term propagation of rodent ssCs is now possible in vitro, and their genetic modification is feasible. However, their differentiation into sperm is possible only under in vivo conditions. Here we show a new in vitro system that can induce full spermatogenesis from ssC lines or any isolated ssCs. The method depends on an organ culture system onto which ssCs are transplanted. The settled ssCs form colonies and differentiate up into sperm. The resultant haploid cells are fertile, and give rise to healthy offspring through micro-insemination. In addition, the system can induce spermatogenesis from ssCs that show spermatogenic failure due to a micro-environmental defect in their original testes. Thus, an in vitro system is established that can be used to correct or manipulate the micro-environmental conditions required for proper spermatogenesis from murine ssC lines.

With the increasing cure rate of paediatric cancers, infertility, as one of the adverse effects of treatments, has become an important concern for patients and their families. Since semen cryopreservation is applicable only for post-pubertal patients, alternative pre-pubertal measures are necessary. Here we demonstrate that testis tissue cryopreservation is a realistic measure for preserving the fertility of an individual. Testis tissues of neonatal mice were cryopreserved either by slow freezing or by vitrification. After thawing, they were cultured on agarose gel and showed spermatogenesis up to sperm formation. Microinsemination was performed with round spermatids and sperm, leading to eight offspring in total. They grew healthily and produced progeny upon natural mating between them. This strategy, the cryopreservation of testis tissues followed by in vitro spermatogenesis, is promising to preserve the fertility of male paediatric cancer patients in the future.

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.

It is known that cells of testis tissues in fetal or neonatal periods have the ability to reconstruct the testicular architecture even after dissociation into single cells. This ability, however, has not been demonstrated effectively in vitro. In our present study, we succeeded in reconstructing seminiferous tubules in vitro which supported spermatogenesis to meiotic phase. Testis cells of neonatal mice were dissociated enzymatically into single cells. The cells formed aggregates in suspension culture and were transferred to the surface of agarose gel to continue the culture with a gas-liquid interphase method, where a tubular architecture gradually developed during the following 2 weeks. Immunohistological examination confirmed Sertoli cells forming tubules and germ cells inside. With testis tissues of Acr-GFP transgenic mice, whose germ cells express GFP during meiosis, cell aggregates formed a tubular structure and showed GFP expressions in their reconstructed tissues. Meiotic figures were also confirmed by regular histology and immunohistochemistry. In addition, we mixed cell lines of spermagonial stem cells (GS cells) into the testis cell suspension, and found the incorporation of GS cells in the tubules in reconstructed tissues. When GS cells derived from Acr-GFP transgenic mice were used, GFP expression was observed, indicating that the spermatogenesis of GS cells was proceeding up to the meiotic phase. This in vitro reconstruction technique will be a useful method for the study of testis organogenesis and spermatogenesis.