Discrimination between HCV29 and T24 by controlled proliferation of cells co-cultured on substrates with different elasticity.

In this work the impact of substrate elasticity on the proliferation of two cell lines, a non-malignant transitional epithelium HCV-29 and a bladder transitional cell carcinoma T24 cultured individually and in co-culture was analyzed. A significantly stronger, highly cell-dependent impact of mechanical properties on cellular behavior was shown for cells co-cultured from the mixture. A more effective proliferation process observed for T24 cells was analyzed quantitatively for asymmetric HCV29: T24 mixtures co-cultured on soft substrate. The obtained results suggest that the proliferation of T24 cells is even 4 times more effective as compared to HCV29 cells and confirm strong invasiveness of metastatic T24 cells. The high adaptiveness of T24 cells to adverse environmental conditions enables easy and accurate discrimination between not isolated healthy and cancer cells.

Precise positioning of cancerous cells on PDMS substrates with gradients of elasticity.

In this work the novel method to create PDMS substrates with continuous and discrete elasticity gradients of different shapes and dimensions over the large areas was introduced. Elastic properties of the sample were traced using force spectroscopy (FS) and quantitative imaging (QI) mode of atomic force microscopy (AFM). Then, fluorescence microscopy was applied to investigate the effect of elastic properties on proliferation of bladder cancer cells (HCV29). Obtained results show that cancerous cells proliferate significantly more effective on soft PDMS, whereas the stiff one is almost cell-repellant. This strong impact of substrate elasticity on cellular behavior is driving force enabling precise positioning of cells.

Protein coverage on silicon surfaces modified with amino-organic films: a study by AFM and angle-resolved XPS.

An approach to determine structural features, such as surface fractional coverage F and thickness d of protein layers immobilized on silicon substrates coated with amino-organic films is presented. To demonstrate the proposed approach rabbit gamma globulins (RgG) are adsorbed from a 0.66muM solution onto SiO(2) and Si(3)N(4) modified with (3-aminopropyl)triethoxysilane (APTES). Atomic force microscopy data are analyzed by applying an integral geometry approach to yield average coverage values for silanized Si(3)N(4) and SiO(2) coated with RgG, F=0.99+/-0.01 and 0.76+/-0.08, respectively. To determine the RgG thickness d from angle-resolved X-ray photoelectron spectroscopy (ARXPS), a model of amino-organic bilayer with non-homogeneous top lamellae is introduced. For an APTES layer thickness of 1.0+/-0.1nm, calculated from independent ARXPS measurements, and for fractional surface RgG coverage determined from AFM analysis, this model yields d=1.0+/-0.2nm for the proteins on both silanized substrates. This value, confirmed by an evaluation (1.0+/-0.2nm) from integral geometry analysis of AFM images, is lower than the RgG thickness expected for monomolecular film ( approximately 4nm). Structures visible in phase contrast AFM micrographs support the suggested sparse molecular packing in the studied RgG layers. XPS data, compared for bulk and adsorbed RgG, suggest preferential localization of oxygen- and nitrogen-containing carbon groups at silanized silicon substrates. These results demonstrate the potential of the developed AFM/ARXPS approach as a method for the evaluation of surface-protein coverage homogeneity and estimation of adsorbed proteins conformation on silane-modified silicon substrates used in bioanalytical applications.