用于睾丸原代细胞及类器官培养的猪睾丸脱细胞支架

Decellularized scaffolds derived from porcine testes for culture of primary testicular cells and organoids

摘要:

脱细胞细胞外基质可完整保留天然组织的仿生超微结构，为细胞与类器官的体外培养提供适配的仿生三维微环境。本研究旨在制备一种完整保留天然超微结构与生物活性成分的猪睾丸脱细胞细胞外基质(porcine testicular decellularized extracellular matrix, PT-dECM)支架，为睾丸原代细胞的黏附增殖及睾丸类器官的体外构建提供高度仿生的三维微环境。本研究优化了物理-化学联合脱细胞方案，经筛选获得了可有效清除免疫原性物质、同时完整保留天然超微结构与生物活性成分的PT-dECM支架。该支架可实现细胞均匀黏附、定向增殖，并为原代睾丸细胞提供了维持结构与功能稳定的三维仿生环境。基于该支架构建的睾丸类器官可呈现天然睾丸的典型生理结构，可稳定检出支持细胞、间质细胞、生殖细胞以及管周肌样细胞这4类睾丸核心功能细胞，其空间分布与天然组织高度一致。本研究优化制备的PT-dECM支架模拟了天然睾丸微环境，弥补了现有模型缺陷，为深化睾丸微环境功能研究提供了新工具，在生殖组织工程领域具有重要的应用前景。

Abstract:

Decellularized extracellular matrix can well preserve the biomimetic ultrastructure of native tissue, providing a suitable three-dimensional biomimetic microenvironment for the in vitro culture of cells and organoids. The aim of this study is to prepare a porcine testicular decellularized extracellular matrix (PT-dECM) scaffold that fully preserves the natural ultrastructure and bioactive components, thereby providing a highly biomimetic three-dimensional microenvironment for the adhesion and proliferation of primary testicular cells and the in vitro construction of testicular organoids. In this study, we optimized a combined physicochemical decellularization protocol and, after screening, obtained a PT-dECM scaffold that could effectively remove immunogenic substances and fully preserve the natural ultrastructure and bioactive components. This scaffold not only supported uniform cell adhesion and directed proliferation but also provided primary testicular cells with a three-dimensional biomimetic environment that maintained structural and functional integrity. Testicular organoids constructed on this scaffold exhibited the typical physiological structure of native testes, and the four core functional cell types—Sertoli cells, Leydig cells, germ cells, and peritubular myoid cells—were reliably detected, with their spatial distribution being highly consistent with that of native tissue. The PT-dECM scaffold optimized in this study mimicked the natural testicular microenvironment, overcoming the limitations of existing models. It provides a new tool for advancing research into the functions of the testicular microenvironment and has significant application potential in the field of reproductive tissue engineering.