EUROPEAN CONGRESS AND EXHIBITION ON ADVANCED MATERIALS AND PROCESSES - EUROMAT 2021

Adherent cell manufacturing is hindered by the necessity to use solid substrates or hydrogels for their culture and expansion. Such materials and platforms are relatively difficult to scale up and parallelise. In contrast, liquid-liquid technologies and microdroplet platforms have been applied very successfully in the field of Chemical Engineering for the scale up of synthesis and purification of fine chemicals, therapeutics, polymers and nanomaterials. Yet, their use in the field of biotechnologies is largely confined to the high throughput screening of planktonic cells. Here we present that the culture of adherent cells at the surface of liquids, previously observed with fibroblasts, is mediated by the self-assembly of protein nanosheets forming mechanically strong interfaces. This enables the adhesion of stem cells such as keratinocytes and mesenchymal stem cells and the regulation of their spreading via the classic integrin and acto-myosin machinery, allowing the retention of stem cell phenotypes despite the extreme compliance of liquid substrates. We report that the viscoelastic behaviour of protein nanosheets correlates with stem cell proliferation. We identify that particularly stiff nanosheets that display poor elasticity do not support stem cell expansion and that this phenomenon is associated with the formation of brittle domains that can relax and dissipate energy in response to cell mediated forces. Hence the multi-scale viscoelasticity of liquid-liquid interfaces, rather than shear moduli, is the primary physical determinant of stem cell proliferation. Stem cells cultured on nanosheet-stabilised emulsions displayed comparable phenotype in long-term expansion compared to stem cells cultured on 2D plastic and solid microcarriers. Overall, our results pave the way to the use of microdroplet technologies and liquid-liquid interfaces in the field of tissue engineering and for stem cell technologies.