Regulation of the Fate of Cells and Cell Ensembles using Tunable Interfaces
Final Report Abstract
The primary goal of the project was the design of physically defined models of tissue surfaces to precisely (i.e. quantitatively) control the morphology and/or development of cells and cell ensembles adhered to them. To achieve this goal, polymer films with different physical properties, as well as biofunctionalized supported lipid membranes, were used as a physical model of the extracellular matrix. Via force-indentation experiments done by atomic force microscopy, it was demonstrated how the modulus of elasticity (Young’s modulus) of DPA-MPC-DPA triblock copolymer films increases linearly with pH between pH 7 and pH 8. The measured range of the Young’s modulus (~ 1 – 40 kPa) covers the preferable elasticity of both muscle cells (~ 10 kPa) and neurons (~ 1 kPa), suggesting a potential of DPA-MPC-DPA films for culturing various cell types. Basing on such values, mouse myoblasts were selected as the test cell system for further experiments, since they live under comparable mechanical conditions in nature. Depending upon the strength of cell adhesion to the surface, cells either spread on the surface, or assume more round shapes. Indeed, after an incubation time of 3 h of the cells on hydrogels at different pH, clearly different spreading behaviors could be observed: the projected area of cells on a stiff hydrogel with E = 40 kPa (600 ± 180 μm2) was about two times larger than that on a soft hydrogel with E = 1.4 kPa (290 ± 50 μm2), indicating that the cells sense the rigidity of the substrate. Supported lipid membranes can easily be biofunctionalized via coupling of engineered proteins on their surface. They are hence ideal candidates to be used as a substrate for tissue culture. In this work, animal cap tissue from the frog Xenopus was placed on supported lipid membranes functionalized with the His-tagged adhesion molecule Xenopus cadherin-11 (XCad-11) and examinated by micro-interferometry. It was found that the number of adhesive cells per unit area increased as a function of time and of XCad-11 density, reaching the maximum value at a lateral distance of the proteins of 5.5 nm, after an incubation time of 4 h. Furthermore, supported lipid membranes can not only employed as substrates for culturing cells, but can be seen themselves as a model of the cell membrane. Artificial supported membranes were deposited on cellulose cushions that had been extensively characterized by X-ray and neutron reflectivity. After deposition of the lipids, the reflectivity curves were consistent with the formation of a homogeneous membrane over a macroscopically large area (cm2 order). The latter was confirmed by fluorescence microscopy. In a further step, X-ray reflectivity was used for the first time to characterize native cell membranes (extracted from erythrocyte ghosts) spread on a cellulose cushion. The obtained results were consistent with a lipid membrane core carrying a glycocalix on the top leaflet and remnants of the cytoskeleton on the bottom leaflet. In future experiments, other types of native membranes (extracted from the sarcoplasmatic reticulum and from HeLa cells) will be deposited on cellulose and characterized in the same fashion.
Publications
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Native supported membranes: creation of two-dimensional cell membranes on polymer supports. BioInterphases, 3, FA 12, 2008
M. Tanaka, F.F. Rossetti, S. Kaufmann
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Structure of regenerated cellulose films revealed by grazing incidence small-angle X-ray scattering. Biointerphases, 3, 117-127, 2008
F.F. Rossetti, P. Panagiotou, F. Rehfeldt, E. Schneck, M. Dommach, S.S. Funari, A. Timmann, P. Müller-Buschbaum, M. Tanaka
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Native supported membranes on planar polymer supports and micro-particle supports. J. Struct. Biol., 168, 2009
M. Tanaka, M. Tutus, S. Kaufmann, F.F. Rossetti, E. Schneck, I. Weiss
