Hydrogels were prepared with 8-arm PEG vinyl sulfone (PEG-VS, 40 kDa, >99% purity, Jenkem Technology) and formed via Michael-type addition with an MMP-sensitive bi-functional crosslinking peptide Ac-GCRDVPMS↓MRGGDRCG (VPMS) (1739 g/mol, >90% purity, GenScript, cleavage site indicated by ↓)74 (link). PEG hydrogels were modified with the integrin binding peptide GC GYGRGDSPG (RGD) (1067.10 g/mol, GenScript) to allow attachment of the encapsulated cells.
Prior to in situ crosslinking and encapsulation, 4% PEG-VS solution was prepared by dissolving PEG in the appropriate amount of 0.05 M HEPES buffer at pH 7.4, and the monofunctional agents (0.75 or 1.5 mM RGD) were allowed to bind to the PEG macromers for 15 min. Meanwhile, ilnFRCs were suspended at a concentration of 2 × 106 cells per ml and centrifuged for 5 min at 300×g to remove excess media. The cell pellet was reconstituted with the modified PEG-VS solution, and the crosslinker (VPMS) was added to form a gel. The stoichiometric ratio of –VS to thiol (–SH) groups was kept constant at 1:1 ratio for all experiments. Each 60-μl gel was crosslinked in a 96-well plate at 37 °C for 30 min and flipped every 5 min to prevent cell sedimentation and clustering. After 30 min, gels were covered with 300 μl of FM-2. Media was changed every 3 days and cells were cultured up to 12 days.
Prior to in situ crosslinking and encapsulation, 4% PEG-VS solution was prepared by dissolving PEG in the appropriate amount of 0.05 M HEPES buffer at pH 7.4, and the monofunctional agents (0.75 or 1.5 mM RGD) were allowed to bind to the PEG macromers for 15 min. Meanwhile, ilnFRCs were suspended at a concentration of 2 × 106 cells per ml and centrifuged for 5 min at 300×g to remove excess media. The cell pellet was reconstituted with the modified PEG-VS solution, and the crosslinker (VPMS) was added to form a gel. The stoichiometric ratio of –VS to thiol (–SH) groups was kept constant at 1:1 ratio for all experiments. Each 60-μl gel was crosslinked in a 96-well plate at 37 °C for 30 min and flipped every 5 min to prevent cell sedimentation and clustering. After 30 min, gels were covered with 300 μl of FM-2. Media was changed every 3 days and cells were cultured up to 12 days.
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