Conventional culture is done on tissue culture polystyrene (TCPS) surfaces that have been treated to become hydrophilic and negatively charged. To improve cell growth and differentiation in vitro, TCPS surface modifications have been proposed including grafting surfaced with recombinant cell adhaesion peptides derived from extracellular matrix (ECM) proteins or using isolated ECM components to coat them. As the ECM represents an alloy of supramolecular assemblies carrying various biologically relevant ligands we hypothesised that such a complex ECM can only be laid down by professional matrix-making cells. Thus, it would be preferable to direct cellular activity rather than to control surface modifications. To this end, we directed EMC-deposition by human fibroblasts by adding negatively charged or neutral macromolecules to the culture medium thus creating a specific biophysical environment characterised by the excluded volume effect. This system allows for a complete conversion of procollagen to collagen by C-proteinase, drives the self-assembly of collagen and stimulates lysyl oxidase-mediated crosslinking of the matrix. As such, more collagen can be deposited in 48 hrs than in 4 weeks or longer, also the differntial usage of different charged macromolecular crowders allows the modulation of the depositional pattern. On this matrix, mesenchymal stem cells proliferated faster and retained their differentiation potential into osteogenic and adipogenic lineages compared to TCPS. Embryonic stem cells remained undifferentiated, formed tighter colonies and exhibited less runaway cells compared to Matrigel. In conclusion, a “dirtier” surface comprising of an ECM that has been bio-assembled under controlled conditions may provide preferable nanotopographic and chemical cues in comparison to artificial single component grafting.

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