Kinetic behaviour of the cells touching substrate: the interfacial stiffness guides cell spreading

Abstract

To describe detailed behaviour of cell spreading under the influence of substrate stiffness, A549 cells cultured on the surfaces of polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) with bulk rigidities ranging from 0.1 kPa to 40 kPa were in situ observed. The spreading behaviour of cells on PAAm presented a positive correlation between spreading speed and substrate stiffness. After computing the deformations of PAAm gels and collagen, the bulk stiffness of PAAm, rather than matrix tethering, determined the cell behaviour. On the other hand, spreading behaviour of the cells was unaffected by varying the bulk stiffness of PDMS. Based on simulation analyses, the elasticity of silica-like layer induced by UV radiation on PDMS surface dominated cell-substrate interaction, rather than the bulk stiffness of the material, indicating that it is the interfacial stiffness that mainly guided the cell spreading. And then the kinetics of cell spreading was for the first time modeled based on absolute rate theory.

Figure 1

(a) Steps of computing cell areas. From left to right: original DIC image, detecting edge, optimizing threshold, filling the hole with removing noise. (b) Time-lapse recording of cell spreading in the first 20 min. (c) Relative contact radius as a function of time. This relationship can be fitted well by the power laws. Scale bar: 10 μm.

Figure 2. Different behaviours of cell spreading on different substrates of PAAm.

(a) Time-lapse recording of cell spreading on different substrates. The extensions of lamellipodia or filopodia rely on substrate elasticity. (b) The projected areas of cells after 20 min seeding. They increase with the elasticity. (c) The speed of cell spreading on different substrates. Cells on stiffer substrate spread much faster than those on soft substrate. ** p < 0.01, obtained by one-way ANOVA analysis. Scale bar: 10 μm.

Figure 3. Independence of cell spreading behaviours on the bulk stiffness of PDMS.

(a) Time-lapse recording of cell spreading on different substrates in the same range of rigidity as PAAm. (b) Nearly the same projected areas, showing no significant statistical differences. (c) Cells spreading fast on both stiff substrate and soft substrate. Scale bar: 10 μm.

Figure 4

(a) Finite element model of indentation simulation composed of indenter and PDMS substrate with surface layer. (b) AFM averaged curve and optimized FE curve.

Figure 5. FEM analysis of the influence of silica-like layer on PDMS.

(a) The PDMS modeled as a half cylinder (150 μm in radius and 60 μm in thickness). Contact area was treated as a semi-circle with the radius of 6 μm, close to the size of focal adhesion. The model comprising a hard surface layer and compliant bulk materials is pulled at a given distance. (b) Shear stress at the surface of bare PDMS on the contact region with meshing buffering region. The contact region (focal adhesion size) was inside the dashed half circle. The maximum value was of the order of 1 kPa. (c–e) Shear stress at the surface of PDMS with hard layer. Owing to the hard layer, the shear stress at the contact area was ten times bigger, of the order of 10 kPa. Changing the bulk stiffness of PDMS from 0.1 kPa (c) to 40 kPa (e) does not contribute much to the shear stress on the surface. Unit in (b–e): MPa.

Figure 6. A simple model using relative deformation of collagen and PAAm gels under cell contraction.

It is established to evaluate the key factor of extracellular matrix tethering and bulk stiffness.

Figure 7. Schematic of cell spreading over a substrate.

The chemical process of actin assembly is affected by the integrin-ECM binding force and membrane resistance.

Figure 8. Normalized contact radius as a function of time.

This result is consistent with the typical data on PAAm in our experiment.