Accurate mechanical characterization of adherent cells and their substrates is important for understanding the influence of mechanical properties on cells themselves. Recent mechanobiology studies outline the importance of mechanical parameters, such as stress relaxation and strain stiffening on the behavior of cells. Numerous techniques exist for probing mechanical properties and it is vital to understand the benefits of each technique and how they relate to each other. This mini review aims to guide the reader through the toolbox of mechanical characterization techniques by presenting well-established and emerging methods currently used to assess mechanical properties of substrates and cells.

Hydrogels are excellent soft materials to interface with biological systems. Precise control and tunability of dissipative properties of gels are particularly interesting in tissue engineering applications. In this work, we produced hydrogels with tunable dissipative properties by photopolymerizing a second polymer within a preformed cross-linked hydrogel network of poly(acrylamide). We explored second networks made with different structures and capacity to hydrogen bond with the first network, namely linear poly(acrylic acid) and branched poly(tannic acid). Gels incorporating a second network made with poly(tannic acid) exhibited excellent stiffness (0.35 ± 0.035 MPa) and toughness (1.64 ± 0.26 MJ m⁻³) compared to the poly(acrylic acid) counterparts. We also demonstrate a strategy to fabricate hydrogels where the dissipation (loss modulus) can be tuned independently from the elasticity (storage modulus) suitable for cell culture applications. We anticipate that this modular design approach for producing hydrogels will have applications in tailored substrates for cell culture studies and in load bearing tissue engineering applications.

In native extracellular matrices (ECM), cells utilize matrix metalloproteinases (MMPs) to degrade and remodel their microenvironment. Accordingly, synthetic matrices have been engineered to permit MMP-mediated cleavage, facilitating cell spreading, migration, and interactions. However, the interplay between matrix degradability and mechanical properties remains underexplored. We hypothesized that MMP activity induces immediate mechanical alterations in the ECM, which are subsequently detected by cells. We observed that both fibrillar collagen and synthetic degradable matrices exhibit enhanced stress relaxation following MMP exposure. Cells responded to these variations in relaxation by modulating their spreading and focal adhesions. Furthermore, we demonstrated that stress relaxation and cell spreading can be precisely controlled through the rational design of matrix degradability. These findings establish a fundamental link between matrix degradability and stress relaxation, with potential implications for a broad spectrum of biological applications.

The extracellular matrix provides complex biophysical cues to cells which respond to these signals with signaling cascades that determine various cellular processes including fate. Many material systems have been explored to mimic the mechanical properties of the extracellular matrix to determine the cell responses to mechanical cues. While stiffness has emerged as an important regulator of cell behavior, recently, other mechanical properties such as strain stiffening and viscoelasticity have also emerged as potent regulators. This review explores the substrates used for studying mechanotransduction and strategies adopted to impart more complex mechanical cues including spatiotemporal control of mechanical properties. In addition, practical considerations for designing hydrogels for cell culture are discussed and the response of cells to viscoelastic cues in particular is discussed in depth. Recent mechanotransduction studies of combinations of mechanical and other cues are finally reviewed. It is anticipated that such multiphysical cues will further the understanding of mechanotransduction involved in complex processes such as migration and mechanical memory and provide a framework in controlling cell behavior.