Measuring the elastic modulus of soft culture surfaces and three-dimensional hydrogels using atomic force microscopy

自愈水凝胶 刚度 原子力显微镜 力谱学 弹性模量 材料科学 生物医学工程 组织工程 软质材料 复合材料 生物系统 纳米技术 计算机科学 高分子化学 工程类 生物
作者
Michael D. A. Norman,Sílvia A. Ferreira,Geraldine M. Jowett,Laurent Bozec,Eileen Gentleman
出处
期刊:Nature Protocols [Nature Portfolio]
卷期号:16 (5): 2418-2449 被引量:107
标识
DOI:10.1038/s41596-021-00495-4
摘要

Growing interest in exploring mechanically mediated biological phenomena has resulted in cell culture substrates and 3D matrices with variable stiffnesses becoming standard tools in biology labs. However, correlating stiffness with biological outcomes and comparing results between research groups is hampered by variability in the methods used to determine Young’s (elastic) modulus, E, and by the inaccessibility of relevant mechanical engineering protocols to most biology labs. Here, we describe a protocol for measuring E of soft 2D surfaces and 3D hydrogels using atomic force microscopy (AFM) force spectroscopy. We provide instructions for preparing hydrogels with and without encapsulated live cells, and provide a method for mounting samples within the AFM. We also provide details on how to calibrate the instrument, and give step-by-step instructions for collecting force-displacement curves in both manual and automatic modes (stiffness mapping). We then provide details on how to apply either the Hertz or the Oliver-Pharr model to calculate E, and give additional instructions to aid the user in plotting data distributions and carrying out statistical analyses. We also provide instructions for inferring differential matrix remodeling activity in hydrogels containing encapsulated single cells or organoids. Our protocol is suitable for probing a range of synthetic and naturally derived polymeric hydrogels such as polyethylene glycol, polyacrylamide, hyaluronic acid, collagen, or Matrigel. Although sample preparation timings will vary, a user with introductory training to AFM will be able to use this protocol to characterize the mechanical properties of two to six soft surfaces or 3D hydrogels in a single day. This protocol describes how to use atomic force microscopy to measure the elastic modulus of soft 2D surfaces and cell-laden 3D hydrogels. We provide instructions for sample preparation, instrument calibration and data collection and analysis.
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