Longo Giovanni, Dinarelli Simone, Collacchi Federica, Girasole Marco
Institute for the Structure of Matter, Italian National Research Council (CNR-ISM), Via del Fosso del Cavaliere 100, 00133 Rome, Italy.
Methods Protoc. 2025 Aug 1;8(4):86. doi: 10.3390/mps8040086.
Erythrocyte (RBC) aging involves significant structural and nanomechanical alterations crucial to their function. This study aims to bridge the gap between analyses based on statistical morphometric parameters, e.g., membrane roughness, and those based on point-dependent nanomechanical properties, e.g., stiffness or Young's modulus. Using Atomic Force Microscopy, we investigated morphology, membrane roughness, and nanomechanical properties on the very same RBCs under dehydrated (air) and hydrated (physiological buffer) conditions. The cells were studied at different stages of in vitro aging: one, seven, and 12 days. Our results quantitatively show that across dehydration, as well as along the aging pathway, RBCs become progressively more rigid while their membrane roughness decreases, a trend observed in both environments. Notably, the differences between the hydrated and dehydrated states were large in young cells but diminished when erythrocytes aged. Despite these parallel trends, high-resolution mapping on the nanoscale revealed that roughness and Young's modulus do not correlate, indicating that these parameters are linked to different properties. In conclusion, this work provides a comprehensive protocol for a biophysical description of RBC aging and establishes that the simultaneous measurement of membrane roughness and nanomechanical properties offers a complementary approach, yielding a more complete characterization of cellular properties.
红细胞（RBC）衰老涉及对其功能至关重要的显著结构和纳米力学改变。本研究旨在弥合基于统计形态计量参数（如膜粗糙度）的分析与基于点相关纳米力学特性（如刚度或杨氏模量）的分析之间的差距。使用原子力显微镜，我们研究了在脱水（空气）和水合（生理缓冲液）条件下同一红细胞的形态、膜粗糙度和纳米力学特性。在体外衰老的不同阶段（1天、7天和12天）对细胞进行了研究。我们的结果定量显示，在脱水过程中以及沿着衰老途径，红细胞逐渐变得更硬，而其膜粗糙度降低，这一趋势在两种环境中均有观察到。值得注意的是，年轻细胞中水合状态和脱水状态之间的差异很大，但随着红细胞衰老，这种差异会减小。尽管有这些平行趋势，但纳米尺度上的高分辨率映射显示粗糙度和杨氏模量不相关，表明这些参数与不同的特性相关。总之，这项工作为红细胞衰老的生物物理描述提供了一个全面的方案，并确定同时测量膜粗糙度和纳米力学特性提供了一种互补方法，能够更全面地表征细胞特性。
