Mechanical response of an epithelial island subject to uniaxial stretch on a hybrid silicone substrate

Abstract

Introduction: The mechanical response of large multi-cellular collectives to external stretch has remained largely unexplored, despite its relevance to normal function and to external challenges faced by some tissues. Here, we introduced a simple hybrid silicone substrate to enable external stretch while providing a physiologically relevant physical micro-environment for cells.

Methods: We micropatterned epithelial islands on the substrate using a stencil to allow for a circular island shape without restraining island edges. We then used traction force microscopy to determine the strain energy and the inter-cellular sheet tension within the island as a function of time after stretch.

Results: While the strain energy stored in the substrate for unstretched cell islands stayed constant over time, a uniaxial 10% stretch resulted in an abrupt increase, followed by sustained increase in the strain energy of the islands over tens of minutes, indicating slower dynamics than for single cells reported previously. The sheet tension at the island mid-line perpendicular to the stretch direction also more than doubled compared to unstretched islands. Interestingly, the sheet tension at the island mid-line parallel to the stretch direction also reached similar levels over tens of minutes indicating the tendency of the island to homogenize its internal stress.

Conclusions: We found that the sheet tension within large epithelial islands depends on its direction relative to that of the stretch initially, but not at longer times. We suggest that the hybrid silicone substrate provides for an accessible substrate for studying the mechanobiology of large epithelial cell islands.

Keywords: mechanobiology; micropatterning; sheet tension; strain; traction force.

Figure 1

Preparation of hybrid silicone substrates and patterning of epithelial cell islands for stretch and traction measurements. Soft silicone was cured on a layer of pre-cured hard PDMS, and then exposed to deep UV light (of wave lengths 185 and 254 nm) (a). It was then incubated with an aqueous mixture containing EDC, sulfo-NHS, fluorescent beads, and collagen I in water (b). After washing with PBS, some cell culture media was added and a perforated stainless steel sheet was placed on the soft silicone sample (c). Cells were then plated in media constrained by a Teflon well (d). After overnight incubation at 37 °C (and 5% CO₂), the Teflon well and perforated sheet were removed and a PDMS well held replaced cell media supplemented with 10 mM HEPES (e).

Figure 2

Application of uniaxial stretch. (a) Schematic of the custom designed uniaxial stretcher. (b) The region of the sample (where the islands are patterned, shown as circles) is schematically shown (middle), with y being the stretch direction. Strain field (ε_yy, ε_xx, ε_xy) of the substrate at the center (right images) and corner (left images) of the sample under 10% uniaxial stretch are shown.

Figure 3

Traction forces exerted by control and uniaxially stretched MDCK islands. (a, b) Traction stress vector field (left) and traction stress magnitude (right) of a micropatterned MDCK cell island that hasn’t been subjected to stretch (a) and that has been subjected to 10% uniaxial stretch (b) for 35–40 min.

Figure 4

Temporal changes in strain energy density stored in the substrate for control (black) and stretched (red) cell islands. Strain energy per unit area of the control cell islands (N = 14 islands) stays essentially constant. Stretched islands (N = 17 islands) exhibit greater strain energy which continues to slowly increase over time after the initial abrupt increase. Error bars show the corresponding values of the standard error of the mean for all islands.

Figure 5

The sheet tension at the midline of control (black) (N = 14 islands) and stretched (red) (N = 17 islands) cell islands. Cell sheet tension of cell islands along the x–x (a) and y–y (b) midlines are shown. Error bars show the corresponding values of the standard error of the mean for all islands.