  It is established that the mechanical properties of hydrogels control the
fate of (stem) cells. However, despite its importance, a one-to-one
correspondence between gels' stiffness and cell behaviour is still missing from
literature. In this work, the viscoelastic properties of Poly(ethylene-glycol)
(PEG)-based hydrogels - broadly used in 3D cell cultures and whose mechanical
properties can be tuned to resemble those of different biological tissues - are
investigated by means of rheological measurements performed at different length
scales. When compared with literature values, the outcomes of this work reveal
that conventional bulk rheology measurements may overestimate the stiffness of
hydrogels by up to an order of magnitude. It is demonstrated that this apparent
stiffening is caused by an induced 'tensional state' of the gel network, due to
the application of a compressional normal force during measurements. Moreover,
it is shown that the actual stiffness of the hydrogels is instead accurately
determined by means of passive-video-particle-tracking (PVPT) microrheology
measurements, which are inherently performed at cells length scales and in
absence of any externally applied force. These results underpin a methodology
for measuring the linear viscoelastic properties of hydrogels that are
representative of the mechanical constraints felt by cells in 3D hydrogel
cultures.
