Probing Cellular Mechanoadaptation Using Cell-Substrate De-Adhesion Dynamics: Experiments and Model

Abstract

Physical properties of the extracellular matrix (ECM) are known to regulate cellular processes ranging from spreading to differentiation, with alterations in cell phenotype closely associated with changes in physical properties of cells themselves. When plated on substrates of varying stiffness, fibroblasts have been shown to exhibit stiffness matching property, wherein cell cortical stiffness increases in proportion to substrate stiffness up to 5 kPa, and subsequently saturates. Similar mechanoadaptation responses have also been observed in other cell types. Trypsin de-adhesion represents a simple experimental framework for probing the contractile mechanics of adherent cells, with de-adhesion timescales shown to scale inversely with cortical stiffness values. In this study, we combine experiments and computation in deciphering the influence of substrate properties in regulating de-adhesion dynamics of adherent cells. We first show that NIH 3T3 fibroblasts cultured on collagen-coated polyacrylamide hydrogels de-adhere faster on stiffer substrates. Using a simple computational model, we qualitatively show how substrate stiffness and cell-substrate bond breakage rate collectively influence de-adhesion timescales, and also obtain analytical expressions of de-adhesion timescales in certain regimes of the parameter space. Finally, by comparing stiffness-dependent experimental and computational de-adhesion responses, we show that faster de-adhesion on stiffer substrates arises due to force-dependent breakage of cell-matrix adhesions. In addition to illustrating the utility of employing trypsin de-adhesion as a biophysical tool for probing mechanoadaptation, our computational results highlight the collective interplay of substrate properties and bond breakage rate in setting de-adhesion timescales.

Bulky glycocalyx drives cancer invasiveness by modulating substrate-specific adhesion

“…4C-E). To further test how MUC1 levels influence adhesion, trypsin de-adhesion assay 28,35 was performed wherein spread cells were incubated with warm trypsin and their detachment kinetics tracked for the duration cells rounded up but remained attached to their substrates (Figs 4F, G). Once again, the de-adhesion time, i.e., the time for cells to round up scaled inversely with MUC1 levels with fastest de-adhesion observed in MUC1-High cells, and neuraminidase treatment abolishing the MUC1-dependent behavior (Fig.…”

“…De-adhesion assay was performed using protocol described elsewhere 27,28 . Briefly, cells grown on collagen/E-selectin coated dishes for 24 hrs were washed with PBS and incubated with warm (37°C) trypsin.…”

High ligand density drives extensive spreading and motility on soft GelMA gels

“…Stability of cell shape is determined by a force balance between the actomyosin cytoskeleton which exerts inward directed contractile force and focal adhesions which resist these forces. We had earlier shown that this balance can be probed using trypsin-induced de-adhesion wherein time kinetics of cell rounding is followed upon incubation of adherent cells with warm trypsin [36,[41][42][43]. To test how tensional homeostasis is achieved on GelMA gels, trypsin de-adhesion assay was performed (figure 3(A)).…”

The effects of substrate morphology by regulating pseudopods formation on cell directional alignment and migration

“…Directional anisotropy and the degree of anisotropy changes over time and spatially which means that the parameters (κ, σ and γ) have a certain relationship with the growth time and space of pseudopod (i.e. the parameters (κ, σ and γ) are functions of time and space) [50]. Therefore, the resistance energy and all the variables presented (κ, σ and γ) as a function of space and time, and the resistance energy in equation ( 12) can be written as:…”