Xeno‐free, chemically defined poly(ethylene glycol) (PEG)‐based hydrogels are being increasingly used for in vitro culture and differentiation of human induced pluripotent stem cells (hiPSCs). These synthetic matrices provide tunable gelation and adaptable material properties crucial for guiding stem cell fate. Here, sequential norbornene‐click chemistries are integrated to form synthetic, dynamically tunable PEG–peptide hydrogels for hiPSCs culture and differentiation. Specifically, hiPSCs are photoencapsulated in thiol–norbornene hydrogels crosslinked by multiarm PEG–norbornene (PEG–NB) and proteaselabile crosslinkers. These matrices are used to evaluate hiPSC growth under the influence of extracellular matrix properties. Tetrazine–norbornene (Tz–NB) click reaction is then employed to dynamically stiffen the cell‐laden hydrogels. Fast reactive Tz and its stable derivative methyltetrazine (mTz) are tethered to multiarm PEG, yielding mono‐functionalized PEG‐Tz, PEG‐mTz, and dualfunctionalized PEG‐Tz/mTz that react with PEG–NB to form additional crosslinks in the cell‐laden hydrogels. The versatility of Tz‐NB stiffening is demonstrated with different Tz‐modified macromers or by intermittent incubation of PEG‐Tz for temporal stiffening. Finally, the Tz–NB‐mediated dynamic stiffening is explored for 4D culture and definitive endoderm differentiation of hiPSCs. Overall, this dynamic hydrogel platform affords exquisite controls of hydrogel crosslinking for serving as a xeno‐free and dynamic stem cell niche.
The development of new material platforms can improve our ability to study biological processes. Here, we developed a water‐compatible variant of a click‐like polymerization between alkynoates and secondary amines to form β‐aminoacrylate synthetic polyethylene glycol (PEG) based hydrogels. These materials are easy to access—PEG alkynoate was synthesized on a 100 gram scale and the amines were available commercially; these materials are also operationally simple to formulate—gel formation occurred upon simple mixing of precursor solutions without the need for initiators, catalysts, nor specialized equipment. Three‐dimensional cell culture experiments also indicated cytocompatibility of these gels with >90 % viability retained in THP‐1 and NIH/3T3 cells after 72 hours in culture. This hydrogel system therefore represents an alternative platform to other click and click‐like hydrogels with improved accessibility and user‐friendliness for biomaterials application.
more » « less- NSF-PAR ID:
- 10079144
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 57
- Issue:
- 49
- ISSN:
- 1433-7851
- Page Range / eLocation ID:
- p. 16026-16029
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract 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.
Basic Protocol 1 : Tuning viscoelasticity by varying alginate molecular weightBasic Protocol 2 : Tuning viscoelasticity with ionic versus covalent crosslinkingBasic Protocol 3 : Tuning viscoelasticity by adding PEG spacers to alginate chainsSupport Protocol 1 : Testing mechanical properties of alginate hydrogelsSupport Protocol 2 : Conjugating cell‐adhesion peptide RGD to alginate -
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Click chemistry reactions have become an important tool for synthesizing user-defined hydrogels consisting of poly(ethylene glycol) (PEG) and bioactive peptides for tissue engineering. However, because click crosslinking proceeds via a step-growth mechanism, multi-arm telechelic precursors are required, which has some disadvantages. Here, we report for the first time that this requirement can be circumvented to create PEG–peptide hydrogels solely from linear precursors through the use of two orthogonal click reactions, the thiol–maleimide Michael addition and thiol–norbornene click reaction. The rapid kinetics of both click reactions allowed for quick formation of norbornene-functionalized PEG–peptide block copolymers via Michael addition, which were subsequently photocrosslinked into hydrogels with a dithiol linker. Characterization and in vitro testing demonstrated that the hydrogels have highly tunable physicochemical properties and excellent cytocompatibility. In addition, stoichiometric control over the crosslinking reaction can be leveraged to leave unreacted norbornene groups in the hydrogel for subsequent hydrogel functionalization via bioorthogonal inverse-electron demand Diels–Alder click reactions with s -tetrazines. After selectively capping norbornene groups in a user-defined region with cysteine, this feature was leveraged for protein patterning. Collectively, these results demonstrate that our novel chemical strategy is a simple and versatile approach to the development of hydrogels for tissue engineering that could be useful for a variety of applications.more » « less
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