Cell seeding

The ACMs were transferred to 96-well plates, and each ACM was placed in an individual well. MSCs were trypsinized, centrifuged, resuspended and injected into the ACMs at multiple sites using a 22-gauge needle at a concentration of 30 × 10⁶/mL to ensure that a sufficient number of cells were injected into the ACMs to construct the engineered corpus cavernosum. The seeded ACMs were then preserved in a 37 °C incubator for 6 hours to allow the cells to aggregate and attach to the ACMs. Next, the seeded ACMs were transferred to 12-well plates containing 3 mL DMEM/F12 supplemented with 10% FBS and 1% penicillin-streptomycin, which were changed daily. The 12-well plates were placed on a shaker spinning at 40 RPM for 14 days in a 37 °C incubator. The seeded ACM samples were examined by haematoxylin-eosin staining using paraffin-embedded sections every 2 days to observe the state of the seeded cells.