Synthetic matrices with dynamic presentation of cell guidance cues are needed for the development of physiologically relevant in vitro tumor models. Towards the goal of mimicking prostate cancer progression and metastasis, we engineered a tunable hyaluronic acid-based hydrogel platform with protease degradable and cell adhesive properties employing bioorthogonal tetrazine ligation with strained alkenes. The synthetic matrix was first fabricated via a slow tetrazine-norbornene reaction, then temporally modified via a diffusion-controlled method using trans-cyclooctene, a fierce dienophile that reacts with tetrazine with an unusually fast rate. The encapsulated DU145 prostate cancer single cells spontaneously formed multicellular tumoroids after 7 days of culture. In situ modification of the synthetic matrix via covalent tagging of cell adhesive RGD peptide induced tumoroid decompaction and the development of cellular protrusions. RGD tagging did not compromise the overall cell viability, nor did it induce cell apoptosis. In response to increased matrix adhesiveness, DU145 cells dynamically loosen cell-cell adhesion and strengthen cell-matrix interactions to promote an invasive phenotype. Characterization of the 3D cultures by immunocytochemistry and gene expression analyses demonstrated that cells invaded into the matrix via a mesenchymal like migration, with upregulation of major mesenchymal markers, and down regulation of epithelial markers. The tumoroids formed cortactin positive invadopodia like structures, indicating active matrix remodeling. Overall, the engineered tumor model can be utilized to identify potential molecular targets and test pharmacological inhibitors, thereby accelerating the design of innovative strategies for cancer therapeutics.
more »
« less
Dynamic Click Hydrogels for Xeno‐Free Culture of Induced Pluripotent Stem Cells
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.
more » « less- NSF-PAR ID:
- 10454649
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Biosystems
- Volume:
- 4
- Issue:
- 11
- ISSN:
- 2366-7478
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
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
-
more » « less
Abstract Organoids are lumen‐containing multicellular structures that recapitulate key features of the organs, and are increasingly used in models of disease, drug testing, and regenerative medicine. Recent work has used 3D culture models to form organoids from human induced pluripotent stem cells (hiPSCs) in reconstituted basement membrane (rBM) matrices. However, rBM matrices offer little control over the microenvironment. More generally, the role of matrix viscoelasticity in directing lumen formation remains unknown. Here, viscoelastic alginate hydrogels with independently tunable stress relaxation (viscoelasticity), stiffness, and arginine–glycine–aspartate (RGD) ligand density are used to study hiPSC morphogenesis in 3D culture. A phase diagram that shows how these properties control hiPSC morphogenesis is reported. Higher RGD density and fast stress relaxation promote hiPSC viability, proliferation, apicobasal polarization, and lumen formation, while slow stress relaxation at low RGD densities triggers hiPSC apoptosis. Notably, hiPSCs maintain pluripotency in alginate hydrogels for much longer times than is reported in rBM matrices. Lumen formation is regulated by actomyosin contractility and is accompanied by translocation of Yes‐associated protein (YAP) from the nucleus to the cytoplasm. The results reveal matrix viscoelasticity as a potent factor regulating stem cell morphogenesis and provide new insights into how engineered biomaterials may be leveraged to build organoids.
-
more » « less
Abstract Photoresponsive hydrogels have become invaluable 3D culture matrices for mimicking aspects of the extracellular matrix. Recent efforts have focused on using ultraviolet (UV) light exposure and multifunctional macromers to induce secondary hydrogel crosslinking and dynamic matrix stiffening in the presence of cells. This contribution reports the design of a novel yet simple dynamic poly(ethylene glycol)–peptide hydrogel system through flavin mononucleotide (FMN) induced di‐tyrosine crosslinking. These di‐tyrosine linkages effectively increase hydrogel crosslinking density and elastic modulus. In addition, the degree of stiffening in hydrogels at a fixed PEG macromer content can be readily tuned by controlling FMN concentration or the number of tyrosine residues built‐in to the peptide linker. Furthermore, tyrosine‐bearing pendant biochemical motifs can be spatial‐temporally patterned in the hydrogel network via controlling light exposure through a photomask. The visible light and FMN‐induced tyrosine dimerization process produces a cytocompatible and physiologically relevant degree of stiffening, as shown by changes of cell morphology and gene expression in pancreatic cancer and stromal cells. This new dynamic hydrogel scheme should be highly desirable for researchers seeking a photoresponsive hydrogel system without complicated chemical synthesis and secondary UV light irradiation.
-
more » « less
Abstract 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.
