Stimuli-responsive Polymer brushes for On-chip Cell adhesion control – SPOC

- We focus on the synthesis of photo-thermoresponsive ter-polymers composed of dimethylacrylamide (DMA), N-isopropylacrylamide (NIPAM) and Azo-acrylamide (AzAAm). These polymers are grown by ATRP (Atom Transfer Radical Polymerization), using a catalyst regeneration technique (ARGET, for Activator ReGenerated by Electron Transfer) that allows us to work in the presence of oxygen. This synthesis pathway is then directly applicable to the growth of polymer brushes on surface by the so-called «grafting-from« method.
- RICM microscopy allows to perform reflectivity measurements on polymer brushes, giving access to their conformation change upon application of a stimulus, and allowing us to deduce their molecular parameters. RICM also allows to perfom studies of cell adhesion (cell imaging, visualization and morphological quantification of focal adhesion patches) with the need to perform functional staining of the cells.

- We succeeded in synthesizeing tep-polymers of DMA-NIPAM-AzAAm showing a LCST that depends on the irradiation history. We have shown that for the propoer molar content of the three monomers, we can induce the reversible collapse/swelling of the polymer upon irradiation at 435/365 nm, at a temperature of 37°C.
- We have developped an original RICM microscope that allows to perform 2D interference imaging at three wavelengths simultaneously, or to perform spectral reflectivity measurements over a range covering the visible spectrum.
- The RICM microscope has been used to characterize PNIPAM brushes in the vicinity of their LCST. We have shown that our optical technique is sensitive enough to reveal an effect of vertical structuring of the brushes when they switch from good to poor solvent conditions. This represents one of the very few experimental evidence of a vertical phase separation effect predicted theoretical several years ago.
- RICM has also been employed to study qualitatively the adhesion of Mouse Embryonic Fibroblasts in the presence of PNIPAM brushes.We have thus been able to clarify the influence of brush grafting density and chain length on cell adhesion/detachment.

The design of substrates allowing for spatiotemporal control of cell adhesion and morphology upon application of an external stimulus is an important challenge. Such substrates represent the next generation of tools that will help to increase our understanding of cell/surface interactions and to study cell adhesion dynamics as well as the mechanistic pathways involved in cells' responses to alterations in their immediate environment.
After succeeding in building the required photo-sensitive polymers, we are currently focusing on the design and characterization of brushes made of such polymers grafted to glass surfaces. Such substrates should allow us to manipulate «on-chip« the adhesive environment of cells, through the control of the local conformation of the brush.

- Probing the conformation of thermo-sensitive polymer brushes with Reflection Interference Contrast Microscopy
S. Varma, L. Bureau, D. Debarre
Oral communication. Condensed Matter Days JMC14/CMD25
24-29 aout 2014, Paris

Controlling and understanding the adhesive interactions between tissue cells and synthetic surfaces is both a fundamental and technological challenge in cell biology. Progresses in material science and soft matter physico-chemistry have allowed designing advanced substrates for in vitro cell culture, as well as tools in order to investigate in always greater details the basic mechanisms controlling cell adhesion. However, questions regarding the dynamics of cell adhesion, or the complex mechano-transduction processes that relates the mechanical properties of a cell’s environment with its fate, remain largely open. While such questions are at the heart of future developments in biomedical and biotechnological applications, the tools to address them are still extremely scarce.
The design of advanced “smart” substrates for cell culture, which adhesive properties can be dynamically altered on-demand, is emerging as a highly promising way to increase our understanding of dynamic cell/surface interactions.
The SPOC project aims at developing smart substrates that exhibit tunable and reversible adhesive properties down to the micrometer scale relevant to single cell processes. By using light as a local stimulus to switch their properties, we expect such substrates to allow for both spatial and temporal control of cell adhesion, and thus to achieve dynamic control of cell shape, adhesion and migration. Our design strategy relies on the synthesis of polymer brushes made of photo-thermo-sensitive polymers that undergo a reversible swelling/collapse transition under light irradiation at the appropriate wavelengths. Such a transition, which is at the basis of cell adhesion modulation on existing thermoresponsive polymer coatings, will be used to create dynamic adhesive patterns allowing to alter “on-chip” the environment of cells and probe the subsequent re-organization in space and time of the focal adhesions that connect the cell cytoskeleton to the extra-cellular matrix. Furthermore, we will perform a quantitative characterization of the mechanical properties of such stimuli-sensitive brushes in order to use our designed patterned substrates as original actuators/force sensors for adhesion forces measurements and cell sorting applications.