8-Arm PEG-DBCO

Guo, Chen, et al. Acta biomaterialia 56 (2017): 80-90.

Researchers demonstrate the use of SPAAC chemistry to control the immobilization and release of bioorthogonal azide-functionalized proteins within hydrogel networks. They developed a method to create layered hydrogels with defined composition using bioorthogonal chemistry; this is the first time SPAAC chemistry has been used to form layered hydrogels. The formation of AzCFP/AzTMBmCh/AzCFP layered gels followed by cleavage of proteins from the middle layer while maintaining the structure of the layered hydrogel.
Using bioorthogonal SPAAC chemistry, by reacting multi-arm PEG functionalized with cyclooctyne groups (8-Arm PEG-DBCO) with azide-bearing proteins and PEG-2-Az,
A hydrogel decorated with intact proteins is formed, covalently cross-linked with multi-arm PEG. 8-Arm PEG-DBCO was synthesized by reacting PEG-8-amine with an excess of DBCO-acid followed by diethyl ether purification, yielding 82 ± 5% DBCO functionality. To ensure complete immobilization of the protein, AzCFP was prereacted with 8-Arm PEG-DBCO overnight and then mixed with the PEG-2-Az cross-linker, which immediately began to form a gel at physiological pH. The relatively small ratio of AzCFP to PEG-2-Az cross-linker will result in lower yields of immobilized protein if these azide groups are mixed simultaneously with 8-Arm PEG-DBCO in the gel-forming solution. Therefore, prereacting 8-Arm PEG-DBCO with azido-based proteins allows for more consistent protein conjugation within the hydrogel structure.

Blatchley, Michael R., et al. Advanced Materials 34.16 (2022): 2109252.

To control the growth and differentiation of organoids in synthetic hydrogels, strain-promoted azide-alkyne cycloaddition (SPAAC) reaction of 8-arm, 20 kDa PEG-dibenzocyclooctyne (PEG-DBCO) and bis-azide functionalized allyl sulfide was used to prepare hydrogels. These hydrogels were further incorporated to promote cell attachment
Starting from single cell suspensions, ISCs were cultured in Matrigel in ENRCV for 3 days. After 3 days, organoid colonies were transferred onto PEG-AlS hydrogels. The hydrogel precursor solution was prepared by diluting 8-arm PEG-DBCO in Advanced DMEM-F12 medium supplemented with GlutaMAX, HEPES and penicillin-streptomycin to 5 wt%. Azide-functionalized RGD (0.8 mM) was added to the hydrogel precursor solution, which was then kept on ice until the addition of cells (20-30 min). Organoids were released from Matrigel using cold medium. Collected organoid colonies were then centrifuged (900 RPM, 4 min) and the pellet resuspended in a hydrogel precursor solution on ice. Laminin (0.2 mg/mL) was then added. Finally, gelation was initiated by the addition of a bis-azide functionalized allyl sulfide crosslinker. Immediately following the addition of the allyl sulfide crosslinker, the solution was vortexed for 5 s and added in 10 μL drops onto coverslips to form gels. The gels were incubated at 37°C for 15-20 min and then incubated in culture medium containing the appropriate supplements as described above (ENRCV), as well as 2.5 μM thiazolylvin (Stemgent) and 1 μM n-acetylcysteine. After two days in culture, organoids were fixed and imaged or subjected to photosoftening. Photosoftening was achieved by incubating organoids in PEG-AlS hydrogels with 15 mM GSH and 1 mM LAP in FluoroBrite medium containing N2 and B27 supplements for 20-30 min at 37°C. The medium was aspirated and the samples were exposed to 365 nm UV light (4.5 mW/cm) for 30 s.

Rao, Varsha V., et al. Acta biomaterialia 145 (2022): 77-87.

The factors secreted by bone marrow mesenchymal stem cells can affect bone homeostasis, including the rate of bone resorption and deposition, which are altered in osteoporosis. Studies have used porous granular hydrogel scaffolds to control the degree of cell aggregation (~20-90%) and cell size clusters (single cell-30 cells/cluster). Increased secretion of activin A, CXCL1, CX3CL1, MCP-1, TIMP-1, and TNF-ɑ was observed for OVX rMSCs in large clusters compared with SHAM rMSCs, indicating that the secretory profile of OVX rMSCs has a pro-absorptive tendency. In addition, fourfold increased expression of N-cadherin was observed in OVX rMSCs compared with SHAM, both when cultured alone and in large clusters. Finally, N-cadherin signaling is partially responsible for regulating the secretory properties of OVX rMSCs. When N-cadherin interaction was blocked, TIMP-1 and MCP-1 secretion was selectively reduced.
Individual microgels were created using PEG-DBCO and PEG-N macromers by reverse phase suspension polymerization in hexane with Span-80 (2.25% v/v) and Tween-20 (0.75% v/v). Two different sets of microgels, one with excess DBCO and the other with excess azide, were prepared with 11mM excess of either functional group in the polymer mixture. The two microgel populations were subsequently mixed to covalently bind the assembled scaffold. For the DBCO excess microgels, 3mM 8-arm PEG-DBCO and 3mM 4-arm PEG-azide were used. 1mM N-GRGDS was present in all microgels. Shear forces were applied by magnetic stirring, vortexing, or sonication during polymerization to produce microgels of various sizes.