制备可调聚丙烯酰胺水凝胶以研究基质硬度对干细胞 Leydig 细胞的影响
Fabricating Tunable Polyacrylamide Hydrogels To Study Matrix Stiffness Effects On Stem Leydig Cells.
摘要(中文译文)
细胞外基质(ECM)硬度对干细胞行为具有关键调控作用。此前，我们已证明衰老过程中基质硬度的病理性增加会破坏干细胞 Leydig 细胞(SLC)稳态，导致睾酮下降。基于这一发现，本文介绍了一套详细的实验方案——最初在本实验室建立——用于制备硬度可调的聚丙烯酰胺(PA)水凝胶，以在体外模拟睾丸微环境。该方法可实现 1–100 kPa 硬度范围内凝胶的可重复制备，覆盖生理至病理条件。关键步骤包括丙烯酰胺/双丙烯酰胺的精确混合、凝胶溶胀平衡、使用 Sulfo-SANPAH 进行表面活化，以及胶原包被以支持 SLC 黏附和培养。我们提供了针对目标硬度的优化配方，以及针对聚合不完全和细胞贴壁不良等常见问题的故障排除指导。该系统可在确定的二维条件下系统研究基底硬度如何调控 SLC 增殖、分化和类固醇生成功能。除生殖生物学外，它还可作为其他细胞类型机械生物学研究以及筛选针对硬度相关功能障碍的治疗药物的有价值平台。
Abstract
Extracellular matrix (ECM) stiffness critically regulates stem cell behavior. Previously, we demonstrated that pathological increases in matrix stiffness during aging disrupt stem Leydig cell (SLC) homeostasis, leading to a decline in testosterone. Building on this discovery, we here present a detailed protocol-originally developed in our laboratory-for fabricating polyacrylamide (PA) hydrogels with tunable stiffness to model the testicular microenvironment in vitro. This method enables reproducible casting of gels across a stiffness range of 1-100 kPa, covering physiological to pathological conditions. Key steps include precise mixing of acrylamide/bis-acrylamide, gel swelling equilibration, surface activation with Sulfo-SANPAH, and collagen coating to support SLC adhesion and culture. We provide optimized formulations for target stiffnesses and troubleshooting guidance for common issues such as incomplete polymerization and poor cell attachment. This system allows systematic investigation of how substrate stiffness modulates SLC proliferation, differentiation, and steroidogenic function under defined 2D conditions. Beyond reproductive biology, it also serves as a valuable platform for mechanobiological studies in other cell types and for screening therapeutics targeting stiffness-related dysfunction.
如何引用
AMA
Moxuan Li, Yixiang Chen, Xiangyu Li, Jiayu Huang, Jiancheng Wang. Fabricating Tunable Polyacrylamide Hydrogels To Study Matrix Stiffness Effects On Stem Leydig Cells.. Journal of visualized experiments : JoVE. 2026; doi:10.3791/71387.
APA
Moxuan Li, Yixiang Chen, Xiangyu Li, Jiayu Huang, Jiancheng Wang (2026). Fabricating Tunable Polyacrylamide Hydrogels To Study Matrix Stiffness Effects On Stem Leydig Cells.. Journal of visualized experiments : JoVE. https://doi.org/10.3791/71387
GB/T 7714
Moxuan Li, Yixiang Chen, Xiangyu Li, Jiayu Huang, Jiancheng Wang. Fabricating Tunable Polyacrylamide Hydrogels To Study Matrix Stiffness Effects On Stem Leydig Cells.[J]. Journal of visualized experiments : JoVE, 2026 doi:10.3791/71387.
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