Profiling native pulmonary basement membrane stiffness using atomic force microscopy

Bastian Hartmann

doi:10.1038/s41596-024-00955-7

Profiling native pulmonary basement membrane stiffness using atomic force microscopy

2024-03-01

1

PRE

AI

Hauke Clausen‐Schaumann *

R

Raphael Reuten *

DOI：10.1038/s41596-024-00955-7

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Abstract

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Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only similar to 100-400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young's modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young's modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young's modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young's moduli and generation of Young's modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1-2 d. The function of pulmonary alveoli is dependent on their mechanical robustness and response to external forces. The Young's modulus (stiffness) of their basement membranes is higher than that of the surrounding cell layers.This protocol describes how to prepare lung sections from humans or mice and perform atomic force microscopy experiments. Challenges in data analysis-including filtering to focus specifically on basement membrane values-are addressed using the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox. Atomic force microscopy can be used to determine the stiffness of materials. This protocol describes how to measure and quantify the Young's modulus E of pulmonary mouse and human basement membranes with atomic force microscopy and the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox.

Keywords:

IMAGING SOFT SAMPLES

ARTICULAR-CARTILAGE

MECHANICAL-PROPERTIES

ELASTIC PROPERTIES

OPTICAL TWEEZERS

GENE-EXPRESSION

SCANNING FORCE

CELL-MIGRATION

MICE LACKING

COLLAGEN I

Profiling native pulmonary basement membrane stiffness using atomic force microscopy

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