The mechanics of liquid-liquid Interfaces controls cell spreading and stem cell expansion

摘要

The expansion and delivery of stem cells to diseased or damaged tissue is an important hurdle to address for the development of regenerative medicine applications. The accepted view in the field of tissue engineering is that adherent cells isolated from their respective tissues need to be grown on rigid substrate and are normally not viable when cultured in suspension or too compliant substrates. However, Trappmann et al showed high cell spreading was observed on soft polydimethylsiloxane (PDMS) substrates (below 1 kPa). Correlated to these observations, stem cell fate was found to be independent of PDMS stiffness over a wide range of compliance (from 1 kPa to 1 MPa). These observations contrast with reports that soft substrates mimic the mechanical properties of specific tissue and enhance the differentiation into defined lineages, such as, stem cells committing to neurogenic differentiation on soft substrates mimicking brain tissue. To explore these conflicting results, this project explores the culture of stem cells at liquid-liquid interfaces. We explore the ability of primary keratinocytes and epidermal cell lines to spread and proliferate on such interfaces using a combination of immunostaining and fluorescence microscopy. We investigate the role of acto-myosin contractility and focal adhesion formation in regulating cell spreading on liquid interfaces. We quantify the mechanical properties of the liquid-liquid interfaces sustain in cell growth using combination of AFM, SEM, Scanning Ion Conductance Microscopy and rheology. Our results show that the nanoscale mechanical behavior of liquid-liquid interfaces allows cell spreading through the typical integrin-actin-myosin based machinery. We find that these interfaces allow the culture of stem cells and the preservation of their potential to differentiate. In addition, we find that, in some conditions, dense cultures of cells can be achieved using liquid-liquid interfaces and that these show typical hallmarks of cell sheets mechanically engaged with their underlying substrates (see Figure 1 showing the actin networks of cell sheets generated on rigid substrates and liquid-liquid interfaces.) These results clearly show that cells do not inherently feel the bulk mechanical properties of substrates, but rather directly sense their mechanical environment at the nanoscale. These findings have important implications for the design of biomaterials for tissue engineering but also the long term expansion of stem cells and the preservation of their regenerative potential. A B Figure 1. Immunofiuorescence image of epidermal cell sheets generated on tissue culture plastic (A) and at a liquid-liquid interface (B). (blue: dapi, red: phalloidin actin).