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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Ahluwalia, Arti (University of Pisa)


Studies investigating cell response to substrate viscoelasticity are usually based on hydrogels where both viscous and elastic properties are altered at the same time [1-2], so the two effects cannot be decoupled. We engineered agarose-based hydrogels with a constant equilibrium elastic modulus and different characteristic relaxation times that can be modulated varying the liquid phase viscosity without modifying the crosslinking of the solid network [2]. The agarose gel properties were optimised for the culture of adipose-derived mesenchymal stem cells (ADSCs) to understand if they are able to sense and respond to viscoelastic substrates with different viscous properties.

0.5% w/v agarose gel were fabricated using aqueous solutions with increasing dextran concentrations (0, 3, 4 % w/v), and hence viscosities. Mechanical properties were investigated using the epsilon-dot method [3]. ADSC (50.000 cells/cm2) were cultured for 7 days on the gels with 2 (high τ) and 4% (low τ) w/v dextran coated with 5% w/v gelatin and in 96-well multiwell plates (τ → ∞) as controls. Bright field images were acquired at day 3 and 7 (Olympus). Then, cells were fixed and stained with DAPI and rhodamine-conjugated phalloidin. Immunostaining was also performed to assess the presence of CD45 (negative marker for cell stemness). Images were acquired with a confocal microscope (Nikon A1, Japan).

The gel instantaneous elastic moduli (Einst) and relaxation time (τ) decreased significantly with increasing dextran concentration, while the equilibrium elastic modulus (Eeq) did not vary significantly. Moreover, Eeq was in the optimal range to mimic the mechanical properties of the stem cell niche (≅ 3 kPa [2]). Bright field imaging at day 3 and 7 allowed us to investigate morphological differences between cells on the two substrates. In the controls, we observed high cell spreading; the formation of round shaped cell clusters and only few elongated cells were noted in the 2% gels. Finally in the 4% gels, we observed higher cell spreading with respect to the 2% gels. The cell area was significantly lower in the gels 2% gels with respect to both the 4% gels and the controls. This suggest that cells are able to sense differences in the substrate viscous behaviour, and that, in the gels with the same elastic behaviour, cell spreading increase with decreasing relaxation time. Moreover, a significantly lower CD45 expression in the gels with respect to the control indicate cell tendence to maintain stemness.

Hydrogel viscoelasticity was engineered allowing the study of cell viscoelastic mechanotransduction as a function of gel viscous behaviour. The design of materials with well-defined relaxation behaviour, able to consider the different time scales involved (cell sensing time, substrate relaxation time and observation time), will be essential for generating biomimetic viscoelastic materials for regenerative medicine applications [3].

1.Yang et al., Nat. Materials, 13,645-652, 2014
2.Cacopardo et al., JMBB, 89,162-167, 2019
3.Cacopardo et al., Tissue Eng. Part B, 2021.


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