2.1. Fibrin gel fabrication

Fibrin gels were fabricated in four steps. First, sterile fibrinogen from human plasma (Millipore Sigma, 341576) was dissolved in sterile PBS (Corning, 21-040-CM) at 80 mg/mL at 37°C for two hours. Second, PEG-NHS (3.4 kDa, SUNBRIGHT DE-034HS, NOF America Corporation) was dissolved in PBS at 8 mg/mL, syringe filtered, and immediately mixed with the dissolved fibrinogen 1:1 by volume (PEG:fibrinogen mole ratio of 10:1). The fibrinogen and PEG were allowed to crosslink for one hour at 37°C. Third, cells were dissociated with Accutase^®, counted, and resuspended in growth media at 4X the desired final cell concentration. The 4X cell and fibrinogen-PEG solutions were mixed 1:1 by volume (PBS control added for cell-free gels) and the mixture was added to the appropriate culture vessel. Fourth, thrombin from human plasma (Millipore Sigma, 605190) was resuspended in cold calcium chloride solution (11.1 mM CaCl₂, 145 mM NaCl, pH 7.4 in DI water) at 20 U/mL. Thrombin solution was added to the cell-fibrinogen-PEG solution 1:1 by volume and quickly mixed by pipetting five times. Gelation occurred at 37°C for 5 minutes before gels were immersed in media or PBS. For cell experiments, media was replenished daily. PS and HS gels were fabricated with final concentrations of 10 mg/mL fibrinogen, 1 mg/mL PEG, 1 U/mL thrombin, 5 mM CaCl₂, and pH 7.4. PS fibrin was fabricated with a final NaCl concentration of 145 mM (physiologic concentration, total chlorine concentration of 155 mM), and HS fibrin was fabricated with a final NaCl concentration of 250 mM (total chlorine concentration of 260 mM). NaCl concentration was adjusted by adding NaCl to the PBS used as the solvent for the fibrinogen and PEG.