Biofabricating Seminiferous Tubules for In Vitro Spermatogenesis

用于体外精子发生的生物制造曲细精管

基本信息

批准号：
10800970
负责人：
Yuguo Lei
金额：
$ 41.38万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-20 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10800970
关键词：
3-Dimensional Acids Affect Alginates Animal Testing Architecture Binding Biological Assay Blood-Testis Barrier Buffers Cell Culture System Cell Culture Techniques Cell Maintenance Cells Chemicals Crosslinker Cultured Cells Custom Diagnosis Diameter Diffusion Edetic Acid Endothelial Cells Epithelium Germ Cells Growth Homing Hormones Human Hydrogels In Vitro Infertility Ions Knock-out Knowledge Male Infertility Mammalian Cell Matrix Metalloproteinases Methods Modeling Mus Patients Peptides Phenotype Physiology Process Production Pump RGD (sequence)Reaction Research Risk Scientist Seminiferous tubule structure Shapes Side Spermatogenesis Spermatogonia Suspension Culture System Technology Testis Time Tissues Toxic effect Tube Tubular formation boys cell growth crosslink divinyl sulfone improved in vivo infertility treatment interstitial interstitial cell male mouse model new technology prepuberty prevent scaffold sertoli cell sperm cell stem cells

项目摘要

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.

项目概要精子发生失败是男性不育的主要原因在体内，精子发生发生在生精器官。该过程由小管周围的间质组织分泌的因子调节。体外睾丸细胞培养系统模拟生精小管结构和精子模型无细胞发育重要的基础研究和开发方法来预防、诊断和治疗不孕症。几十年来，科学家们开发了各种睾丸细胞培养系统，包括2D培养和3D细胞然而，有或没有支架悬浮支持的体外精子发生（IVS）的培养。曲细精管的组织尚未在体外成功建立，因为其结构是。对于精子发生至关重要，完整的精子发生周期尚未在体外有效实现。该项目建议使用先进的微挤压技术来精确制造微管体内曲细精管和周围间质组织的细胞组成和组织。为了方便起见，它被称为生物制造的生精小管（BioST）。假设 IVS 可能是这样的。通过建立类似体内的微环境有效地实现了该研究的基础。 PI 实验室的先进细胞培养技术以及 Co- 数十年的精子发生研究 PI 的实验室开发了一种名为 AlgTubes 的哺乳动物细胞培养新技术。悬浮在细胞培养基中的微型藻酸盐水凝胶管中的细胞微型管提供细胞。具有与生理相关的微环境，从而显着提高培养效率，例如 AlgTubes 与生精小管有许多相似之处，例如它们的活力、生长速度和产量。该项目将研究管状形状、微尺度直径以及通过管壁扩散的质量传输。重新利用并进一步开发 AlgTubes 来构建 BioST，其具有三个具体目标：制造 BioST、描述 BioST 的特征，包括其结构、血睾屏障、精原干细胞维持、三个月的雄性激素产生和体外精子发生，并通过建模验证 BioST 先前研究的 Premel1 基因敲除小鼠模型的研究结果对于我们来说非常有价值。它也可以作为动物试验的替代方案。这将减少动物试验的使用并提供更准确和相关的结果。结果，因为体外精子将来自人类细胞，而且，青春期前的男孩正在经历这种情况。性腺毒性治疗有耗尽体外衍生的精原干细胞（SSC）的风险。同样，精子可以为这些男孩提供解决方案，该技术将有利于不孕症的治疗。不能产生精子但仍具有 SSC 的非梗阻性无精症患者。