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Abstract

Cells and tissues are constantly exposed to mechanical forces. Understanding how these forces act on cells to regulate essential processes like development, differentiation, tissue homeostasis and how their alteration is related to disease requires the characterization of their mechanical properties. Several methods have been developed to study mechanical properties of cells and nuclei. However, most of the established methods are not com- patible with culturing of cells in 3D substrates, a factor which plays an essential role in defining the structural and mechanical behavior of cells naturally existing in 3D environ- ments. In this work, image and model based methods have been developed to approach this problem and enable the characterization of the cells mechanical phenotype in 3D.

On a first step, a previously developed method to measure the compressibility of the nuclear interior was enhanced to enable statistical significant measurements of nuclei to perform comparative analyses between phenotypes. Optimization of both the experi- mental, as well as the image processing methods led to a robust framework that served to measure an increase in nuclear compressibility in nuclei of LMNA−/− mouse embryonic fibroblasts. This study served as a proof of principle for this contact free method, which in a subsequent step was adapted to work for cells embedded in 3D substrates.

Aiming to develop a method, in which specific forces could be applied and relate to cellular deformations, the second part of this work was centered in the development of the 3D substrate stretcher. This involved identifying and implementing the needs of the experimental and image analysis framework to ensure the required environment for the cells, while at the same time enabling the acquisition of suitable data for the mechanical analysis. The resulting experimental and analysis framework enables for the first time application and quantification of strains on cells embedded in 3D substrates.

Motivation of the 3D-culture based methods was the analysis of epithelial-mesenchymal transition (EMT) in hepatocytes. These epithelial cells undergoing dedifferentiation upon treatment with TGF-β serve not only as a preeminent example of the need of 3D cell cultures in the characterization of mechanical properties, but also as a model of malignant transformation in fibrotic diseases and cancer. Quantification of previously unobserved morphological and structural properties led to the mechanical phenotyping of these cells, where a decrease in the compressibility of the nuclear interior, an enhanced resistance to deformation and a better anchorage of the nuclei inside the cells was observed after EMT.