Exploiting the superior protein resistance of polymer brushes to control single cell adhesion and polarisation at the micron scale

Abstract

The control of the cell microenvironment on model patterned substrates allows the systematic study of cell biology in well defined conditions, potentially using automated systems. The extreme protein resistance of poly(oligo(ethylene glycol methacrylate)) (POEGMA) brushes is exploited to achieve high fidelity patterning of single cells. These coatings can be patterned by soft lithography on large areas (a microscope slide) and scale (substrates were typically prepared in batches of 200). The present protocol relies on the adsorption of extra-cellular matrix (ECM) proteins on unprotected areas using simple incubation and washing steps. The stability of POEGMA brushes, as examined via ellipsometry and SPR, is found to be excellent, both during storage and cell culture. The impact of substrate treatment, brush thickness and incubation protocol on ECM deposition, both for ultra-thin gold and glass substrates, is investigated via fluorescence microscopy and AFM. Optimised conditions result in high quality ECM patterns at the micron scale, even on glass substrates, that are suitable for controlling cell spreading and polarisation. These patterns are compatible with state-of-the-art technologies (fluorescence microscopy, FRET) used for live cell imaging. This technology, combined with single cell analysis methods, provides a platform for exploring the mechanisms that regulate cell behaviour.

Cell-Extracellular Matrix Interactions in Normal and Diseased Skin

“…Studies with cultured human epidermal cells have delineated some of the signaling pathways involved (Watt 2002), revealing a key role for Erk MAPK (MAP kinase) signaling downstream from b1 integrins in providing a differentiation-inhibitory signal (Zhu et al 1999;Levy et al 2000;Haase et al 2001;Evans et al 2003). Consistent with the differentiation of epidermal cells in suspension, restricting the spreading of single cells on micropatterned substrates stimulates terminal differentiation (Watt et al 1988;Gautrot et al 2010). However, in this case the signal is not dependent on the composition or concentration of the ECM coating, or on focal adhesion formation.…”

Shushing down the epigenetic landscape towards stem cell differentiation

“…By contrast, the shape that cells were constrained to occupy strongly influenced their differentiation state. These effects seemed to be mediated through changes in the cytoskeleton (Gautrot et al, 2010). It will be interesting to further understand this phenomenon, as it is likely to be a general feature of stem cells.…”

“…Her work included a collaborative effort with bioengineers to grow epidermal stem cells on surfaces that had distinct extracellular matrices printed in patterns to dictate the shape in which each cell grew (Gautrot et al, 2010). Their goal was to determine how physical constraints modified a cell's behavior.…”

"Chameleon" macromolecules: synthesis, structures and applications of stimulus responsive polymers

“…62,63 An important issue with respect to possible biomedical applications is the long-term stability of grafts grown from these initiators. 64 The degradation and possible cleavage of polymer grafts may have detrimental Chapter 3 32 consequences for the properties. 65,66 For instance, the stability and non-fouling properties of poly(poly(ethylene glycol) methacrylate) grafts prepared using trimethoxysilane-based anchoring under cell-culture conditions were discussed by Tugulu et al 66 It was shown that at high chain densities, these grafts rapidly detached from the modified surface.…”

“…[62][63][64][65][66] PNIPAM layers with a surface gradient in grafting densities can be prepared by varying the initiator coverage across the grafted substrate. 67 This "high throughput" approach allowed the imaging of the morphology variation at one substrate in the different grafting regimes.…”