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Additional Supporting Information may be found in the online version of this article.

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DVDY_22161_sm_suppinfoFigS1.tif1739KSupp. Fig. 1. Uniluminal vascular spheroid fusion involves disruption of the interfacial disc. Shown are LSCM optical sections of two uniluminal spheroids within the 3–8 hr of hanging drop culture. The fusing spheroids were immunolabeled with antibodies to SMαA (red) and PECAM (green). The image highlights a disruption of the SMαA-expressing cell layers (asterisks) in the interfacial disc region. Also shown is an apparent fusion of the endothelial layers (arrow) of the adjacent spheroids in the space created by the disruption of the SMαA-expressing cell layers. Bars = 50 μm (A) and 25 μm (B).
DVDY_22161_sm_suppinfoFigS2.tif924KSupp. Fig. 2. The process of uniluminal vascular spheroid fusion is dependent on protein synthesis. Uniluminal vascular spheroids were treated with culture medium supplemented with 0.02% DMSO, or culture medium supplemented with 20 μg/ml cycloheximide/0.02% DMSO for 2 hr prior to being placed in hang drop culture in pairs. Following the 2-hr treatment period, spheroids were cultured in new hanging drops consisting of culture medium alone. A, B: Representative pairs of DMSO and cyclohexamide/DMSO-treated uniluminal vascular spheroids, respectively, after 5 hr in hanging drop culture. C, D: The resulting fusion of the pairs of uniluminal vascular spheroids shown in A and B after 18 hr in hanging drop culture. Bars equal 100 μm.
DVDY_22161_sm_suppinfoFigS3.tif992KSupp. Fig. 3. Models of vascular fusion. A: The fusion of two spheroids, each having a central spherical cavity. B: Two adjacent blood vessels arranged in a parallel configuration fusing to form a single, larger diameter blood vessel. At points along the adjacent blood vessels, the curved margins (dashed lines) contact to form interface planes similar to those observed in the fusion of two uniluminal spheroids and the paired dorsal aortae. C: How the joining of a luminized vascular sprout to a preexisting blood vessel might be analogous to the process of uniluminal vascular spheroid fusion in which curved margins at the tip of a sprout (dashed lines) might fuse to the curved margin of an existing blood vessel. The interface of the sprout tip and the adjacent blood vessel would then correspond to the interface plane as seen in the fusion of two uniluminal spheroids.
DVDY_22161_sm_suppinfoFigS4.tif774KSupp. Fig. 4. The process of uniluminal vascular spheroid fusion is scalable allowing formation of larger diameter uniluminal vascular spheroids. A: A light microscopic image of 5 uniluminal spheroids within the first hour of hanging drop culture. B: The resulting composite uniluminal spheroid within 8–12 hr of hanging drop culture. C: LSCM optical sections of the composite uniluminal vascular spheroid shown in B immunolabeled with antibodies to SMαA (red) and PECAM (green). Bars = 100 μm.

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