Cell sheet technology meets macromolecular crowding: The self-ssembly approach for corneal stromal development
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Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop extracellular matrix-rich implantable devices. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of neutral macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 hours in culture, even at low serum concentration. The addition of negative charged galactose derivative (carrageenan) in human corneal fibroblast culture, even at 0.5% serum, increases by 12-fold tissue-specific matrix deposition, whilst maintaining physiological cell morphology and protein / gene expression. Gene analysis indicates that a glucose derivative (dextran sulphate) may drive corneal fibroblasts towards a myofibroblast lineage. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture, with morphological and histological properties similar to native tissue. Collectively, these results indicate that macromolecular crowding may be suitable not only for clinical translation and commercialisation of tissue engineering by self-assembly therapies, but also for the development of in vitro pathophysiology models.