2.2. Hydrogel Preparation

Hydrogels were produced from vinylsulfone functionalized 8-arm PEG star macromers (PEG-VS) with a molecular weight of 40 kDa. All polymers were purchased from JenKem Technology USA. Polymers were dissolved either in PL or in serum-free alpha minimum essential media (α-MEM, Sigma-Aldrich, catalog no. 32561). All hydrogels contained 5 wt % polymer. An MMP-cleavable linking peptide (Ac-GCRDVPMSMRGGDRCG-NH₂) synthesized by Pepmic and dissolved in deionized water (50 mM) was used to end-link the macromers into gels. The same MMP-cleavable linking peptide was used for all hydrogels produced as part of this study. Three sets of gels were generated for this study: PL-loaded (PL-PEG), SDF-1α (SDF1α-PEG)-loaded, and serum-free α-MEM (MEM-PEG) PEG-VS hydrogels. Only MEM-PEG and SDF1α-PEG gels were functionalized with 2.5 mM cRGD [Cyclo(RGD(dF)C), AnaSpec, Fremont, CA]. All SDF1α-PEG gels were loaded with 250 ng/mL SDF-1α. No RGD was added to the PL-PEG gels. PL-PEG gels contained 90 vol % PL solution and 10 vol % of end-linker solution. To produce uniformly shaped gel disks, polymer–end-linker mixtures were pipetted between two glass slides (coated with Sigmacote, Sigma-Aldrich) separated using a 1 mm spacer and allowed to react at 37 °C for 30 min. For migration studies, 65 μL of the gel mixture was pipetted directly into the inlets of the chemotaxis μ-chamber slides (ibidi, Germany). Finally, for cell invasion studies, 30 μL of hydrogels was formed into disks with encapsulated cell spheroids.