Figure 2
From: An approach to quantifying 3D responses of cells to extreme strain
Cellular responses in strained μMASTs.
(a) Confocal fluorescence images of cells in μMASTs after 3 days of straining to different levels (Green:F-actin (phalloidin); blue: nuclei (DAPI)). (b) Mean cell spreading area increased with culture time and with strain level, up to a threshold. In all cases, cell spreading increased nonlinearly with increasing strain, rising rapidly to an asymptotic saturation level at a critical strain in the range of 20–30%. In 10 kPa synthetic tissues, the rise in mean cell spreading area was nearly an order of magnitude, 5 times greater than for the more compliant synthetic tissues. (c) Polarization of NIH/3T3 cells in strained μMASTs. For 6 kPa μMASTs, cells oriented perpendicular to the applied stretch at lower strain levels and parallel to the applied stretch (90°) at higher strain levels. At the lowest and highest levels of stretch, no dominant polarization was observed. For 2 and 10 kPa μMASTs, cell orientation with the direction of stretch was evident at intermediate levels of strain. (d) The number of cells per μMAST increased with time and up to a threshold (10% strain for 2 kPa μMASTs, 20% strain for 6 kPa μMASTs and 30% strain for 10 kPa μMASTs, respectively), with different strain levels. (e,f) Quantification of live, apoptotic and late apoptotic (dead) cells in μMASTs as a function of straining showed a substantial drop in cell viability in the range of 100–150% strain. Cells in μMASTs were analyzed for apoptosis using Annexin V/FITC and PI staining and flow cytometry after 5 days of straining to prescribed levels. Live cells: Q3 (Annexin V(−)/PI(−)); early apoptotic cells: Q4 (Annexin V(+)/PI(−)); late apoptotic cells: Q2 (Annexin V(+)/PI(+)). Control group: cells cultured in μMASTs without straining. Synthetic tissue modulus: 6 kPa. Error bars, s.d. (b–d: 10 ≤ n ≤ 15 μMASTs for each strain level; e,f: 60 ≤ n ≤ 80 μMASTs for each strain level, **p < 0.01, ***p < 0.001). Scale bars: 50 μm.
