Atomic force microscopy of 3T3 and SW-13 cell lines: an investigation of cell elasticity changes due to fixation

Mechanical properties of single cells are of increasing interest both from a fundamental cell biological perspective and in the context of disease diagnostics. In this respect, atomic force microscopy (AFM) has become a powerful tool for imaging and assessing mechanical properties of biological samples. However, while these tests are typically carried out on chemically fixed cells, the most important data is that on living cells. The present study applies AFM technique to assess the Young\u2019s modulus of two cells lines: mouse embryonic fibroblasts (NIH/3T3) and human epithelial cancer cells (SW-13). Both living cells and those fixed with paraformaldehyde were investigated. This analysis quantifies the difference between Young\u2019s modulus for these two conditions and provides a coefficient to relate them. Knowing the relation between Young modulus of living and fixed cells, allows carrying out and comparing data obtained during steady-state measurements on fixed cells that are more frequently available in the clinical and research settings and simpler to maintain and probe

Influence of different fixation reagents on NIH 3T3 fibroblasts morphology: An AFM study

The Atomic force microscope (AFM) has recently become an attractive tool for biological analysis, especially for the characterization of both morphological and mechanical properties. In this study, we investigated the influence of different fixation reagents on NIH 3T3 fibroblast cells morphology. Three protocols were selected: paraformaldehyde, methanol and acetone. Height, perimeter, area, volume and roughness of cells were measured using an AFM. As expected, different fixation protocols produce different morphological changes in cells. However, to preserve fibroblastsi\u301 morphology, the methods applying aldehyde fixations performed much better than those using either methanol or acetone. Based on quantitative assessments, fibroblasts fixed with paraformaldehyde showed the closest volume, area and perimeter values to living cells. Roughness data and a Western blot test confirmed that methanol fixation produces larger cell membrane damage, when compared to the other two fixatives

Metabolic and proliferative cells activity on different substrates

Scaffolds for tissue engineering can be either natural or synthetic materials. The latter allow control of chemical, physical and mechanical properties and also provide support and shape, however they are not of biological origin, and therefore could not promote cell adhesion. This problem does not occur in natural materials however, they have the drawback of having non-suitable mechanical properties and tend to deteriorate too fast. In this work we study the influence of different substrate on the metabolic as well as proliferative cells activity. In particular 4 substrates have been considered: (i) medical grade StageFlexer (silicone elastomer), (ii) Polydimethylsiloxane (PDMS), (iii) PDMS with a layer of carbon nanotubes (CNT\u2019s) and (iv) PDMS with a layer of ceramic whiskers. The results of both tests (metabolic and proliferative capacity) showed that the PDMS without any surface treatment, is the worst of the tested substrates. The reason is to be found in the fact that the PDMS is highly hydrophobic and therefore cells have low adhesion to the substrate. This represents a major limitation of PDMS and its functionalization is necessary to improve cell adhesion. Cells placed on PDMS samples with CNT\u2019s show higher metabolic activity and proliferative capacity, compared to the PDMS and PDMS treated with fibronectin. However, the best outcomes have occurred with the PDMS substrate coated with ceramic whiskers