Abstract
The culture of adherent cells is overwhelmingly relying on the use of solid substrates to support cell adhesion. Indeed, it is typically thought that relatively strong bulk mechanical properties (bulk moduli in the range of kPa to GPa) are essential to promote cell adhesion and, in turn, regulate cell expansion and fate decision. In this report, we show that adherent stem cells such as mesenchymal stem cells and primary keratinocytes can be cultured at the surface of liquid substrates and that this phenomenon is mediated by the assembly of polymer nanosheets at the liquid-liquid interface. We use interfacial rheology to quantify this assembly and demonstrate the strong mechanical properties of such nanosheets. Importantly, we show that cell adhesion to such quasi-2D materials is mediated by the classical integrin/acto-myosin machinery, despite the absence of bulk mechanical properties of the underlying liquid substrate. Finally, we show that stem cell proliferation and fate decision are also regulated by the mechanical properties of these self-assembled protein nanosheets. Liquid substrates offer attractive features for the culture of adherent cells and stem cells, and the development of novel stem cell technologies, such as liquid-liquid systems, are particularly well-adapted to automated parallel processing and scale up.
Highlights
S olid substrates displaying bulk moduli in the range of kPa to GPa are typically used for the culture of adherent cells and stem cells and as scaffolds for tissue engineering
We demonstrate that these interfaces promote stem cell expansion at liquid−liquid interfaces, at comparable rates to those observed on solid substrates, and that the nanoscale mechanics of these interfaces regulates cell fate
Our results demonstrate that the nanoscale mechanical properties of materials can regulate stem cell phenotype and expansion independently of bulk mechanical properties
Summary
S olid substrates displaying bulk moduli in the range of kPa (soft hydrogels and elastomers) to GPa (tissue culture polystyrene, TPS, or hydroxyapatite) are typically used for the culture of adherent cells and stem cells and as scaffolds for tissue engineering. Considering the importance of substrate mechanics on cell phenotype, cell culture at the surface of low-viscosity liquids has been observed, mainly in the context of fibroblasts[20−22] and myoblasts.[23] This phenomenon has been studied with fluorinated oils, such as the fluorinated ether Novec 7500, and poly(dimethylsiloxane) In such systems, the use of surfactant molecules was reported to play an important role in the regulation of cell proliferation, the underlying mechanism was not known.[22] Recently, we presented evidence for the formation of mechanically strong protein nanosheets self-assembling at the interfaces between cell culture medium and the supporting oil.[24,25] We observed a direct correlation between the concentration of the prosurfactant pentafluorobenzoyl chloride (PFBC) and the interfacial shear modulus of interfaces to which bovine serum albumin adsorbed. We demonstrate the proof-ofconcept of using emulsions for stem cell expansion and their simple transfer to other substrates
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