Discrimination between NSIP- and IPF-Derived Fibroblasts Based on Multi-Parameter Characterization of Their Growth, Morphology and Physic-Chemical Properties

Abstract

Background: The aim of the research presented here was to find a set of parameters enabling discrimination between three types of fibroblasts, i.e., healthy ones and those derived from two disorders mimicking each other: idiopathic pulmonary fibrosis (IPF), and nonspecific interstitial pneumonia (NSIP). Methods: The morphology and growth of cells were traced using fluorescence microscopy and analyzed quantitatively using cell proliferation and substrate cytotoxicity indices. The viability of cells was recorded using MTS assays, and their stiffness was examined using atomic force microscopy (AFM) working in force spectroscopy (FS) mode. To enhance any possible difference in the examined parameters, experiments were performed with cells cultured on substrates of different elasticities. Moreover, the chemical composition of cells was determined using time-of-flight secondary ion mass spectrometry (ToF-SIMS), combined with sophisticated analytical tools, i.e., Multivariate Curve Resolution (MCR) and Principal Component Analysis (PCA). Results: The obtained results demonstrate that discrimination between cell lines derived from healthy and diseased patients is possible based on the analysis of the growth of cells, as well as their physical and chemical properties. In turn, the comparative analysis of the cellular response to altered stiffness of the substrates enables the identification of each cell line, including distinguishing between IPF- and NSIP-derived fibroblasts.

Cell-Specific Response of NSIP- and IPF-Derived Fibroblasts to the Modification of the Elasticity, Biological Properties, and 3D Architecture of the Substrate

“…Further, the stiffness of the fibroblasts was determined using AFM-based force spectroscopy for indentation depths equal to 200 and 600 nm, providing information from different cell areas [ 80 , 81 , 82 ]. Measurements were made for cells cultured on collagen-covered soft and stiff PDMS substrates, and compared with the mechanical properties of cells cultured on uncoated PDMS [ 56 ]. The relative change in cellular stiffness, determined for the soft and stiff PDMS substrates, is presented in Figure 3 a,b, respectively.…”

“…The fibrotic process that occurs in the progression of ILDs is related to the reorganization of the cytoskeleton architecture and results in altered mechanical properties of disordered cells compared to healthy ones, as well as in different responses to the altered stiffness of the substrate [ 56 ]. However, the presence of ECM proteins, especially collagen, can additionally influence the properties and functions of lung fibroblasts, modifying also their interactions with substrates of different elasticities.…”

“…In turn, disorders in the biochemical composition of the lungs, especially pathological accumulation of collagen and cholesterol [ 26 , 28 , 45 , 46 ] lead to increased tissue stiffness and influence the contractile and proliferative functions of lung fibroblasts, which can result in the transformation of fibroblasts into myofibroblasts [ 24 , 27 , 36 , 39 , 47 , 48 , 49 , 50 , 51 , 52 , 53 ]. The impact of matrix elasticity on the development of IPF has already been examined [ 48 , 52 , 54 , 55 ], though analogous research for NSIP-derived cells is very rare [ 56 ]. Changes in the cytoskeleton organization observed for IPF and NSIP-derived fibroblasts result in differences in their mechanical properties [ 57 ], which leads to modifications of their stiffness in response to altered substrate elasticity [ 56 ].…”

“…The impact of matrix elasticity on the development of IPF has already been examined [ 48 , 52 , 54 , 55 ], though analogous research for NSIP-derived cells is very rare [ 56 ]. Changes in the cytoskeleton organization observed for IPF and NSIP-derived fibroblasts result in differences in their mechanical properties [ 57 ], which leads to modifications of their stiffness in response to altered substrate elasticity [ 56 ].…”