Adherent cultures of each cell type were detached from the culture vessels with 0.25% trypsin-EDTA (Life Technologies, Grand Island, NY) solution. Trypsin was neutralized with appropriate GM, and cells were counted by a hemocytometer. Each cell type was then diluted to a concentration of 2500, 5000, and 10,000 cells in 200 μl of appropriate GM. The cell suspension (200 μl) was then pipetted into a single well of a U-bottom 96-well microplate with a cell-repellent surface (Greiner Bio-One, Monroe, NC). For fabrication of MSC/HUVEC spheroids made of 50,000 cells, MSCs and HUVECs were combined at a ratio of 92:8. The microplates were then incubated at 37°C in a 5% CO2 humidified atmosphere. Spheroid formation was monitored daily on an EVOS FL cell imaging system (Life Technologies). For fabrication of GFP+ HDF/MSC coculture spheroids, 5000 cells were used in total, and GFP+ HDFs and MSCs were cocultured in ratios of 2:1 and 1:1 for 1 day. During the fabrication and culture of HUVECs spheroids, EGM-2MV medium (Lonza) was used.

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