Recently, it has been recognized that natural extracellular matrix (ECM) and tissues are viscoelastic, while only elastic properties have been investigated in the past. How the viscoelastic matrix regulates stem cell patterning is critical for cell‐ECM mechano‐transduction. Here, this study fabricated different methacrylated hyaluronic acid (HA) hydrogels using covalent cross–linking, consisting of two gels with similar elasticity (stiffness) but different viscoelasticity, and two gels with similar viscoelasticity but different elasticity (stiffness). Meanwhile, a second set of dual network hydrogels are fabricated containing both covalent and coordinated cross–links. Human spinal cord organoid (hSCO) patterning in HA hydrogels and co‐culture with isogenic human blood vessel organoids (hBVOs) are investigated. The viscoelastic hydrogels promote regional hSCO patterning compared to the elastic hydrogels. More viscoelastic hydrogels can promote dorsal marker expression, while softer hydrogels result in higher interneuron marker expression. The effects of viscoelastic properties of the hydrogels become more dominant than the stiffness effects in the co‐culture of hSCOs and hBVOs. In addition, more viscoelastic hydrogels can lead to more Yes‐associated protein nuclear translocation, revealing the mechanism of cell‐ECM mechano‐transduction. This research provides insights into viscoelastic behaviors of the hydrogels during human organoid patterning with ECM‐mimicking in vitro microenvironments for applications in regenerative medicine.
Physical properties of the extracellular matrix (ECM) affect cell behaviors ranging from cell adhesion and migration to differentiation and gene expression, a process known as mechanotransduction. While most studies have focused on the impact of ECM stiffness, using linearly elastic materials such as polyacrylamide gels as cell culture substrates, biological tissues and ECMs are viscoelastic, which means they exhibit time‐dependent mechanical responses and dissipate mechanical energy. Recent studies have revealed ECM viscoelasticity, independent of stiffness, as a critical physical parameter regulating cellular processes. These studies have used biomaterials with tunable viscoelasticity as cell‐culture substrates, with alginate hydrogels being one of the most commonly used systems. Here, we detail the protocols for three approaches to modulating viscoelasticity in alginate hydrogels for 2D and 3D cell culture studies, as well as the testing of their mechanical properties. Viscoelasticity in alginate hydrogels can be tuned by varying the molecular weight of the alginate polymer, changing the type of crosslinker—ionic versus covalent—or by grafting short poly(ethylene‐glycol) (PEG) chains to the alginate polymer. As these approaches are based on commercially available products and simple chemistries, these protocols should be accessible for scientists in the cell biology and bioengineering communities. © 2021 Wiley Periodicals LLC.
- Award ID(s):
- 1846367
- PAR ID:
- 10238595
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 1
- Issue:
- 5
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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