(318e) In Situ Manipulation of Microenvironment Modulus to Examine Its Influence On Cell Fate

Physical properties of the cell microenvironment have become increasingly recognized as regulators of cellular functions^{1, 2} such as migration,³ cytoskeletal organization,⁴ and differentiation.⁵ In particular, microenvironment elasticity, as measured by modulus, has been shown to affect cell phenotype. For example, different discrete moduli samples have been used to examine the influence of elasticity on stem cell differentiation⁵ and the fibroblast-to-myofibroblast transition.⁶ While advances have been made in the fabrication of materials for cell culture, the properties of these material systems typically are fixed upon formation. In situ control of material properties is needed to study dynamic cellular processes such as differentiation. Here, we utilize a cell culture platform that allows in situ manipulation of the hydrogel properties with light to study the effect of dynamic changes in modulus on cell fate. Specifically, dynamic substrates were employed to mediate the fibroblast-to-myofibroblast transition, and the fate of myofibroblasts that have been de-activated by tuning the cell microenvironment modulus was explored.

The fibroblast-to-myofibroblast transition is important for understanding wound healing and tissue fibrosis. In response to microenvironment signals such as TGF-Β1 or modulus, fibroblasts are known to activate into myofibroblasts, secreting extracellular matrix proteins and exhibiting a highly organized cytoskeleton with Α-smooth muscle actin (ΑSMA) stress fibers, a conclusive indicator of myofibroblastic differentiation.⁶ The photodegradable hydrogel culture platform⁷ allows precise de-activation of these cells with modulus alone,⁸ but the fate of these de-activated myofibroblasts is unknown. To explore the fate de-activated valvular interstitial cells (VICs, fibroblasts that comprise the heart valve), VICs were cultured on photodegradable hydrogels and their activation and fate was controlled through in situ modulus tuning with light. The photodegradable hydrogel culture platform was formed by copolymerization of a photodegradable PEG-based diacrylate crosslinking monomer with a monoacrylate PEG monomer and contains an entrapped adhesion protein, fibronectin, to promote cell attachment. Upon irradiation of the photodegradable hydrogel under cytocompatible conditions (365 nm at 10 mW/cm² for up to 5 min), the modulus of the cell microenvironment was lowered through degradation of the photolabile PEG crosslinks, de-activating the myofibroblasts and allowing evaluation of their ultimate fate. It was hypothesized that these de-activated cells would become (i) apoptotic, (ii) quiescent, and (iii) senescent. To test this hypothesis, VICs were cultured on myofibroblast promoting substrates (32 kPa) and irradiated, decreasing the gel modulus in situ (7 kPa) and de-differentiating the VICs from myofibroblasts to fibroblasts. De-activation was assessed by immunostaining for ΑSMA stress fibers and RT-PCR for myofibroblast gene expression, including ΑSMA and SMAD-7. Apoptosis was assayed by examining Annexin-V expression using flow cytometry. Cell quiescence was assessed by evaluating cell re-activation into myofibroblasts in response to TGF-Β1 with immunostaining, RT-PCR, and real-time tracking of cytoskeletal organization. Photodegradable hydrogels are a unique dynamic cell culture platform for exploring how real-time changes in cell-material interactions influence dynamic cellular processes such as differentiation and ultimately fate.

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