Biofabricating Seminiferous Tubules for In Vitro Spermatogenesis

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Project Summary Failed spermatogenesis is a major cause of male infertility. In vivo, spermatogenesis occurs in seminiferous tubules. The process is regulated by factors secreted by the interstitial tissue surrounding the tubules. An in vitro testis cell culture system mimics the seminiferous tubule architecture and models spermatogenesis would significantly advance fundamental studies and developing methods to prevent, diagnose, and treat infertility. In past decades, scientists have developed various testis cell culture systems, including 2D culture and 3D cell suspension culture with or without scaffold support for in vitro spermatogenesis (IVS). However, the cellular organization of seminiferous tubules has not been successfully established in vitro. Because the architecture is critical for spermatogenesis, the complete spermatogenesis cycle has not been efficiently achieved in vitro. This project proposes using advanced micro-extrusion technology to precisely fabricate microtubes with cellular composition and organization of the in vivo seminiferous tubules and surrounding interstitial tissue. For convenience, it is termed bio-fabricated seminiferous tubules (BioSTs). The hypothesis is that IVS could be efficiently achieved by establishing an in-vivo-like microenvironment. The proposed research is based on advanced cell culture technologies from the PI lab and decades of studies on spermatogenesis from the Co- PI’s lab. The PI has developed a new technology called AlgTubes for mammalian cell culture. AlgTubes culture cells in micro-scale alginate hydrogel tubes suspended in a cell culture medium. The microtubes provide cells with a physiology-relevant microenvironment, thus, significantly improving the culture efficiency such as viability, growth rate, and yield. AlgTubes have many similarities to the seminiferous tubules, such as their tubular shape, micro-scale diameter, and mass transport via diffusion through the tube wall. This project will repurpose and further develop AlgTubes to build BioSTs with three specific aims: to fabricate BioSTs, to characterize BioSTs including their architecture, blood-testis-barrier, spermatogonia stem cells maintenance, male hormone production, and in vitro spermatogenesis for three months, and to validate BioSTs by modeling findings from a previously studied mouse model with Premel1 knockout. BioSTs will be invaluable for advancing the knowledge of spermatogenesis and male infertility. It also can be an alternative to animal testing in high-throughput toxicity assays. This will reduce animal testing use and provide more accurate and relevant results, as the in-vitro sperm will be derived from human cells. Moreover, prepubertal boys undergoing gonadotoxic treatments are at risk for depleting their spermatogonial stem cells (SSCs). In-vitro-derived spermatozoa can provide a solution for those boys. Similarly, the technology will benefit the infertility treatment of non-obstructive azoospermia patients who cannot produce spermatozoa but still have SSCs.