C7983 50ea

The method of tissue digestion was the same as that of the single-cell sample preparation, the difference was that the digestion time for ECs was 20 min, while that of FB cells was about 40 min. The digested single-cell suspensions were washed and re-suspended in 10% FBS/DMED (Gibco) and EGM-2 (CC-3202; Lonza) medium for FB and EC culturing, respectively. Because of the strong proliferation capacity of FBs, FB cells would eventually dominate in DMEM medium, and the purity was more than 95% after passage. For ECs, cell cloning began at about day 5 of culture, then fibroblast inhibitors were added for 7–10 days. When isolated colonies began to fuse with each other, the EC colonies were marked and digested using a clonal cylinder (C7983-50EA; Sigma-Aldrich), and the EC suspension was further purified using CD31 magnetic beads (130-091-935; Miltenyi).

Fresh CC tissue samples of approximately 1 × 1 cm size were first immersed in 15 mL PBS with vigorous shaking to remove residual blood cells. Then, tissues were cut into pieces of 1 × 1 mm size and enzymatically digested with 2.5 mg/mL collagenase type I (17104-019, gibco, US), 4 mg/mL collagenase type IV (17018029, gibco), 0.1 mg/mL neutral protease, and 2 mg/mL DNase I (#AMPD1, Sigma-Aldrich, US) at 37 °C for 20 min. Subsequently, the cell suspension was filtered through a 40 mm nylon mesh, and the cells were sorted by MACS with a Dead Cell Removal Kit (130-090-101, Miltenyi Biotec, Germany) to remove dead cells. Briefly, 10⁶ cells were co-incubated with 20 µL MicroBeads at 4 °C for 30 min. After blocking and washing, magnetic separation with LS Columns (130-042-401; Miltenyi) and MidiMACS Separator (130-042-302; Miltenyi) was performed, retaining cells that were not adsorbed by the column. CCECs were cultured with EGM-2 medium and cell cloning began at approximately day 5 of culture, then fibroblast inhibitors were added for 7–10 days. When isolated colonies began to fuse with each other, the EC colonies were marked and digested using a clonal cylinder (C7983-50EA; Sigma-Aldrich), and the EC suspension could be further purified using CD31 magnetic beads (according to their purity) (130-091-935; Miltenyi).