Figure 2.

Layered‐hypoxic hydrogels enable uniform ECFC cluster behavior, and match conventional hypoxic hydrogel cluster formation kinetics and O₂. A,B) ECFCs were encapsulated in conventional hypoxic hydrogels (A) and in the layered‐hypoxic hydrogels (B). 40 min after encapsulation, uniform cell seeding was confirmed in both conditions, as measured by images taken at three distinct z‐planes (z1 [lowest] → z3 [highest]). After 24 h in culture, clusters formed in both hydrogels. In the conventional hypoxic hydrogel, clusters were in focus at z1, with no cells in focus at z2 but cells in single cell morphology at z3, indicating non‐uniform cell behavior. In the layered‐hypoxic hydrogels, uniform cell behavior was observed, with all cells in focus at z1, and no cells in focus at z2 or z3. C,D) O₂ measurements show rapid exposure to hypoxia (<5% O₂, grayed area in inset) in both conditions and exposure to hypoxia over the entire 24 h culture period. All scale bars: 50 µm.