A total of 1000 2-day cultured spheroids of each type (with ~2500 cells per spheroid) were collected and homogenized in 100 μl of distilled water. Then, 100 μl of cell homogenate was transferred to a 2-ml pressure-tight vial, added with 100 μl of 12 M hydrochloric acid, and hydrolyzed at 120°C for 3 hours. The total collagen amount per spheroid type was quantified using a hydroxyproline colorimetric assay kit (BioVision Inc., CA) according to the manufacturer’s instructions. Collagen amount expressed by each spheroid type was determined using a PowerWave X-340 spectrophotometer (BioTek, Winooski, VT) at 560 nm, and the results were normalized to the collagen amount expressed by HUVEC spheroids at day 2. Experiments were repeated four times.

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