Biofabricating Seminiferous Tubules for In Vitro Spermatogenesis

项目摘要 精子发生失败是男性不育的主要原因。在体内，精子发生在生精细胞中 小管。这个过程是由小管周围的间质组织分泌的因子调节的。An In 体外睾丸细胞培养系统模拟生精小管的结构和模型 大力推进预防、诊断和治疗不孕不育的基础研究和开发方法。在……里面 在过去的几十年里，科学家们开发了各种睾丸细胞培养系统，包括2D培养和3D细胞 有或没有支架支架的悬浮培养用于体外精子发生(IVS)。然而，蜂窝 生精小管的组织在体外尚未成功建立。因为这个架构是 精子发生是精子发生的关键，在体外还没有有效地实现完整的精子发生周期。 该项目提出使用先进的微挤压技术来精确制造微管 体内生精小管和周围间质组织的细胞组成和组织。为 为了方便起见，它被称为生物制造生精小管(BioSTs)。假说是IVS可能是 通过建立一个类似体内的微环境来有效地实现。拟议的研究是基于 来自PI实验室的先进细胞培养技术和数十年来关于精子发生的研究- 派的实验室。国际和平研究所开发了一种用于哺乳动物细胞培养的新技术，名为海藻管。藻管培养 悬浮在细胞培养液中的微米级藻酸盐水凝胶管中的细胞。微管提供细胞 具有与生理相关的微环境，从而显著提高培养效率，如 生存能力、生长速度和产量。藻管与生精小管有许多相似之处，例如它们的 管状形状、微尺度直径和通过管壁扩散的质量传输。这个项目将 重新利用和进一步发展海藻管来建造BioSTs，有三个具体的目标：制造BioSTs， BioSTs的特征包括其结构、血-睾丸屏障、精原干细胞的维持、 三个月的雄性激素产生和体外精子发生，并通过建模验证BioSTs 之前研究过的带有Premel1基因敲除的小鼠模型的研究结果。BioSTs将对 提高对精子发生与男性不育的认识。它也可以作为动物试验的替代方法。 在高通量毒性测试中。这将减少动物试验的使用，并提供更准确和相关的 结果，由于体外精子将来自人类细胞。此外，青春期前的男孩正在接受 性腺毒性治疗有耗尽精原干细胞(SSCs)的风险。体外衍生 精子可以为这些男孩提供解决方案。同样，这项技术将有利于不孕不育的治疗 不能产生精子但仍有SSCs的非梗阻性无精子症患者。

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.