At predetermined time points (24 hours), media changes were accompanied by supplemental addition of mTG (0.6 to 2.4 U/ml). Rheological analysis (AR1500ex; TA Instruments) was performed as described above using an immersion cup. We assume uniform secondary cross-linking by the addition of supplemental mTG. While diffusion of mTG into the existing matrix potentially leads to a nonuniform matrix stiffness, we assume uniformity through a measurement of bulk matrix properties. ECFCs encapsulated in Gel-HI were monitored up to day 3 by capturing bright-field images (Olympus IX50), at which point they were fixed and stained with phalloidin and DAPI as described above. Vascular networks and lumen were observed using confocal microscopy (LSM 780; Zeiss). Networks with and without the addition of mTG were analyzed.

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