Reliable, standardized measurements for cell mechanical properties.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation.

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (µm). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young's Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with µm spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells. In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer's guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

Mechanical alterations of the hippocampus in the APP/PS1 Alzheimer's disease mouse model.

There is increasing evidence of altered tissue mechanics in neurodegeneration. However, due to difficulties in mechanical testing procedures and the complexity of the brain, there is still little consensus on the role of mechanics in the onset and progression of neurodegenerative diseases. In the case of Alzheimer's disease (AD), magnetic resonance elastography (MRE) studies have indicated viscoelastic differences in the brain tissue of AD patients and healthy controls. However, there is a lack of viscoelastic data from contact mechanical testing at higher spatial resolution. Therefore, we report viscoelastic maps of the hippocampus obtained by a dynamic indentation on brain slices from the APP/PS1 mouse model where individual brain regions are resolved. A comparison of viscoelastic parameters shows that regions in the hippocampus of the APP/PS1 mice are significantly stiffer than wild-type (WT) mice and have increased viscous dissipation. Furthermore, indentation mapping at the cellular scale directly on the plaques and their surroundings did not show local alterations in stiffness although overall mechanical heterogeneity of the tissue was high (SD∼40%).

Viscoelastic properties of white and gray matter-derived microglia differentiate upon treatment with lipopolysaccharide but not upon treatment with myelin.

BACKGROUND: The biomechanical properties of the brain have increasingly been shown to relate to brain pathology in neurological diseases, including multiple sclerosis (MS). Inflammation and demyelination in MS induce significant changes in brain stiffness which can be linked to the relative abundance of glial cells in lesions. We hypothesize that the biomechanical, in addition to biochemical, properties of white (WM) and gray matter (GM)-derived microglia may contribute to the differential microglial phenotypes as seen in MS WM and GM lesions. METHODS: Primary glial cultures from WM or GM of rat adult brains were treated with either lipopolysaccharide (LPS), myelin, or myelin+LPS for 24 h or left untreated as a control. After treatment, microglial cells were indented using dynamic indentation to determine the storage and loss moduli reflecting cell elasticity and cell viscosity, respectively, and subsequently fixed for immunocytochemical analysis. In parallel, gene expression of inflammatory-related genes were measured using semi-quantitative RT-PCR. Finally, phagocytosis of myelin was determined as well as F-actin visualized to study the cytoskeletal changes. RESULTS: WM-derived microglia were significantly more elastic and more viscous than microglia derived from GM. This heterogeneity in microglia biomechanical properties was also apparent when treated with LPS when WM-derived microglia decreased cell elasticity and viscosity, and GM-derived microglia increased elasticity and viscosity. The increase in elasticity and viscosity observed in GM-derived microglia was accompanied by an increase in Tnfα mRNA and reorganization of F-actin which was absent in WM-derived microglia. In contrast, when treated with myelin, both WM- and GM-derived microglia phagocytose myelin decrease their elasticity and viscosity. CONCLUSIONS: In demyelinating conditions, when myelin debris is phagocytized, as in MS lesions, it is likely that the observed differences in WM- versus GM-derived microglia biomechanics are mainly due to a difference in response to inflammation, rather than to the event of demyelination itself. Thus, the differential biomechanical properties of WM and GM microglia may add to their differential biochemical properties which depend on inflammation present in WM and GM lesions of MS patients.

Viscoelastic mapping of mouse brain tissue: Relation to structure and age.

There is growing evidence that mechanical factors affect brain functioning. However, brain components responsible for regulating the physiological mechanical environment are not completely understood. To determine the relationship between structure and stiffness of brain tissue, we performed high-resolution viscoelastic mapping by dynamic indentation of the hippocampus and the cerebellum of juvenile mice brains, and quantified relative area covered by neurons (NeuN-staining), axons (neurofilament NN18-staining), astrocytes (GFAP-staining), myelin (MBP-staining) and nuclei (Hoechst-staining) of juvenile and adult mouse brain slices. Results show that brain subregions have distinct viscoelastic parameters. In gray matter (GM) regions, the storage modulus correlates negatively with the relative area of nuclei and neurons, and positively with astrocytes. The storage modulus also correlates negatively with the relative area of myelin and axons (high cell density regions are excluded). Furthermore, adult brain regions are ∼ 20%-150% stiffer than the comparable juvenile regions which coincide with increase in astrocyte GFAP-staining. Several linear regression models are examined to predict the mechanical properties of the brain tissue based on (immuno)histochemical stainings.

Dynamic indentation reveals differential viscoelastic properties of white matter versus gray matter-derived astrocytes upon treatment with lipopolysaccharide.

Astrocytes in white matter (WM) and gray matter (GM) brain regions have been reported to have different morphology and function. Previous single cell biomechanical studies have not differentiated between WM- and GM-derived samples. In this study, we explored the local viscoelastic properties of isolated astrocytes and show that astrocytes from rat brain WM-enriched areas are ~1.8 times softer than astrocytes from GM-enriched areas. Upon treatment with pro-inflammatory lipopolysaccharide, GM-derived astrocytes become significantly softer in the nuclear and the cytoplasmic regions, where the F-actin network appears rearranged, whereas WM-derived astrocytes preserve their initial mechanical features and show no alteration in the F-actin cytoskeletal network. We hypothesize that the flexibility in biomechanical properties of GM-derived astrocytes may contribute to promote regeneration of the brain under neuroinflammatory conditions.

In vivo characterization of chick embryo mesoderm by optical coherence tomography-assisted microindentation.

Embryos are growing organisms with highly heterogeneous properties in space and time. Understanding the mechanical properties is a crucial prerequisite for the investigation of morphogenesis. During the last 10 years, new techniques have been developed to evaluate the mechanical properties of biological tissues in vivo. To address this need, we employed a new instrument that, via the combination of micro-indentation with Optical Coherence Tomography (OCT), allows us to determine both, the spatial distribution of mechanical properties of chick embryos, and the structural changes in real-time. We report here the stiffness measurements on the live chicken embryo, from the mesenchymal tailbud to the epithelialized somites. The storage modulus of the mesoderm increases from (176 ± 18) Pa in the tail to (716 ± 117) Pa in the somitic region (mean ± SEM, n = 12). The midline has a mean storage modulus of (947 ± 111) Pa in the caudal (PSM) presomitic mesoderm (mean ± SEM, n = 12), indicating a stiff rod along the body axis, which thereby mechanically supports the surrounding tissue. The difference in stiffness between midline and presomitic mesoderm decreases as the mesoderm forms somites. This study provides an efficient method for the biomechanical characterization of soft biological tissues in vivo and shows that the mechanical properties strongly relate to different morphological features of the investigated regions.

Micro-indentation and optical coherence tomography for the mechanical characterization of embryos: Experimental setup and measurements on chicken embryos.

The investigation of the mechanical properties of embryos is expected to provide valuable information on the phenomenology of morphogenesis. It is thus believed that, by mapping the viscoelastic features of an embryo at different stages of growth, it may be possible to shed light on the role of mechanics in embryonic development. To contribute to this field, we present a new instrument that can determine spatiotemporal distributions of mechanical properties of embryos over a wide area and with unprecedented accuracy. The method relies on combining ferrule-top micro-indentation, which provides local measurements of viscoelasticity, with Optical Coherence Tomography, which can reveal changes in tissue morphology and help the user identify the indentation point. To prove the working principle, we have collected viscoelasticity maps of fixed and live HH11-HH12 chicken embryos. Our study shows that the instrument can reveal correlations between tissue morphology and mechanical behavior. STATEMENT OF SIGNIFICANCE: Local mechanical properties of soft biological tissue play a crucial role in several biological processes, including cell differentiation, cell migration, and body formation; therefore, measuring tissue properties at high resolution is of great interest in biology and tissue engineering. To provide an efficient method for the biomechanical characterization of soft biological tissues, we introduce a new tool in which the combination of non-invasive Optical Coherence Tomography imaging and depth-controlled indentation measurements allows one to map the viscoelastic properties of biological tissue and investigate correlations between local mechanical features and tissue morphology with unprecedented resolution.

Regional variations in stiffness in live mouse brain tissue determined by depth-controlled indentation mapping.

The mechanical properties of brain tissue play a pivotal role in neurodevelopment and neurological disorders. Yet, at present, there is no consensus on how the different structural parts of the tissue contribute to its stiffness variations. Here, we have gathered depth-controlled indentation viscoelasticity maps of the hippocampus of acute horizontal live mouse brain slices. Our results confirm the highly viscoelestic nature of brain tissue. We further show that the mechanical properties are non-uniform and at least related to differences in morphological composition. Interestingly, areas with higher nuclear density appear to be softer than areas with lower nuclear density.