(520f) Stiffening Hydrogels to Study Human Lung Fibroblast Activation and Mechanical Memory

The composition and structure of the extracellular matrix (ECM) is a critical regulator of cell function, particularly in response to injury and disease. For example, during lung fibrosis the normally soft and compliant viscoelastic tissue gradually stiffens as resident fibroblasts activate and produce excessive amounts of ECM. This drives fibrogenesis and eventually leads to organ failure. In vitro cell culture platforms provide a pathway to investigate cell-microenvironment interactions, with a recent shift from using supraphysiologically stiff tissue culture plastic (TCP) toward biomaterials, and specifically hydrogels, which provide a more accurate model of the native tissue environment. Hydrogels allow for the presentation of specific mechanical, structural, and adhesive signals to study how these cues affect cell function. We describe a viscoelastic hydrogel system comprised of methacrylated hyaluronic acid (HA) crosslinked through a base-catalyzed thiol-Michael type click reaction to model the Young’s modulus of compliant lung tissue (~ 1.5 kPa). Addition of cyclodextrin-modified HA allows for incorporation of guest-host supramolecular interactions to produce a hydrogel with time-dependent stress relaxation properties, much like native lung tissue. Exposure to blue light (400-500 nm) for a defined length of time allows secondary crosslinking of excess methacrylates to stiffnesses mimicking early (~ 6 kPa) and late (> 30 kPa) stages of lung fibrosis, as characterized through nanoindentation measurements (Figure 1 A, B). Importantly, the secondary photocrosslinking is cytocompatible and can be performed in the presence of cells. Human lung fibroblasts (HLFs) seeded atop either static (1.5 kPa, 6 kPa, >30 kPa) (Figure 1C) or dynamic (1.5 - 6 kPa, 1.5 - >30 kPa, 6 - >30 kPa) materials display distinct cell spreading and shape behaviors as well as differences in focal adhesion organization and chromatin condensation (Figure 1D), depending on the stiffness as well as time length of initial priming. Further, early (< 3) or late (> 10) passage HLF response to this hydrogel system highlight the confounding effects of mechanical priming from TCP culture in subsequent cell studies on hydrogels, likely attributed to cellular mechanical memory. Ongoing work aims to evaluate primary mouse lung fibroblast response with either no, minimal (< 2 passages), or extended (> 5 passages) initial TCP culture in response to this hydrogel system.