Protein Nanosheet Mechanics Controls Cell Adhesion and Expansion on Low-Viscosity Liquids

Abstract

Adherent cell culture typically requires cell spreading at the surface of solid substrates to sustain the formation of stable focal adhesions and assembly of a contractile cytoskeleton. However, a few reports have demonstrated that cell culture is possible on liquid substrates such as silicone and fluorinated oils, even displaying very low viscosities (0.77 cSt). Such behavior is surprising as low viscosity liquids are thought to relax much too fast (<ms) to enable the stabilization of focal adhesions (with lifetimes on the order of minutes to hours). Here we show that cell spreading and proliferation at the surface of low viscosity liquids are enabled by the self-assembly of mechanically strong protein nanosheets at these interfaces. We propose that this phenomenon results from the denaturation of globular proteins, such as albumin, in combination with the coupling of surfactant molecules to the resulting protein nanosheets. We use interfacial rheology and atomic force microscopy indentation to characterize the mechanical properties of protein nanosheets and associated liquid-liquid interfaces. We identify a direct relationship between interfacial mechanics and the association of surfactant molecules with proteins and polymers assembled at liquid-liquid interfaces. In addition, our data indicate that cells primarily sense in-plane mechanical properties of interfaces, rather than relying on surface tension to sustain spreading, as in the spreading of water striders. These findings demonstrate that bulk and nanoscale mechanical properties may be designed independently, to provide structure and regulate cell phenotype, therefore calling for a paradigm shift for the design of biomaterials in regenerative medicine.

Engineering the cellular mechanical microenvironment – from bulk mechanics to the nanoscale

“…F ad , adhesion force; F n and F t , forces applied by the cell in the normal and tangential directions, respectively. Reproduced with permission from Kong et al (2018a) where it was published under a Creative Commons Attribution license (CC BY 4.0). (E) HPKs cultured on fibronectin (Fn)-coated tissue culture plates (TPS-Fn, top) and protein nanoshets, assembled at either pH 7 (soft; middle) or pH 10 (stiff; bottom).…”

“…Cells are even able to form stable adhesions and spread on substrates that lack bulk mechanical stability, such as the surface of liquids. The culture of cells on liquid-liquid interfaces has been demonstrated through the formation of a mechanically stable protein nanosheet (15-20 nm in thickness) at the interface (Kong et al, 2018a(Kong et al, ,b, 2017. These nanosheets are formed via pro-surfactantassisted self-assembly of proteins, and they enable cells to form mature FAs and to spread and to proliferate on the surface of liquid substrates (e.g.…”

Modulation of Mesenchymal Stem Cells Mechanosensing at Fluid Interfaces by Tailored Self‐Assembled Protein Monolayers

“…At the interface between PFC and culture medium, protein film presumably forms as a rigid skin to which cells attach and spread. In these studies, however, an addition of crosslinking agents, such as, glutaraldehyde to the aqueous phase,[12a] reactive surfactants in the organic/oil phase, or the use of charge–charge interactions between protein and polylysine, was indispensable for cell spreading and proliferation. The free‐standing protein film situated at the interface substantially lacks vertical constraint and mechanical properties associated with bulk materials.…”

Materials Nanoarchitectonics as Cell Regulators

“…Gautrot and co-workers reported the control of cell adhesion and expansion by protein nanosheet on low-viscosity liquids. [28] With addition of reactive surfactant pentafluorobenzoyl chloride, the mechanics of protein layer is enhanced, which can promote cell adhesion and proliferation. The same research team reported expansion and fate decision of stem cells with self-assembled nanosheets.…”