ECFC encapsulation, morphogenesis, and network formation in HA gels (days 0-2). A. Vacuole formation observed a few hours after cell encapsulation using: (i) Light microscopy (LM) imaging (left panel) and higher magnification of vacuolated cells (indicated by arrowheads; right panel); (ii) vacuole vital stain FM 4-64 (cyan; nuclei in blue) of encapsulated cells and higher magnification of a vacuolated cell (indicated by arrowheads; inset). Scale bars are 100 μm. (iii) TEM high-resolution representative image of a single, rounded encapsulated cell. Scale bar is 10 μm. (B) Increased number and size of vacuoles and merging into large lumen detected by day 1 of culture, as indicated by: (i) LM imaging (left panel) and higher magnification showing merging lumen (indicated by the arrows; right panel); (ii) vacuole vital stain FM 4-64 (cyan; nuclei in blue) high magnification focused on cells containing large vacuoles (indicated by the arrowhead; scale bars are 50 μm); and (iii) TEM high-resolution representative image of an encapsulated cell with apparent vacuoles. N indicates nucleus; V, vacuoles; L, lumen; and H, hydrogels. Scale bar is 20 μm. (C) Progression in tubulogenesis through branching and sprouting and hydrogel degradation observed using: (i) LM imaging of encapsulated cells on day 2 (left panel) and higher magnification focusing on branching networks (arrows indicate an example; right panel; scale bars are 100 μm); (ii) vacuole vital stain FM 4-64 (cyan; nuclei in blue) on day 2 illustrating representative images of (a) network formation, (b) branching cell, and (c) sprouting cell (scale bars are 50 μm); and (iii) TEM high-resolution representative images of: (a) a cell with degraded surroundings; (b) elongated cell morphology; and (c) guiding channels of degraded hydrogel formed between adjacent cells (indicated by arrows). N indicates nucleus; V, vacuoles; L, lumen; and H, hydrogel. Scale bars are 20 μm.