Engineered Tissue Raft Generation

The in vitro engineered tissue raft was generated as previously described with modifications [27 (link)]. Briefly, 24-mm tissue culture inserts for six-well plates (Transwell Permeable Supports, Corning) were coated with 60% of bovine collagen I (Organogenesis) in DMEM / 10% FBS as an acellular layer in the bottom followed by incubation at 37°C for 1 hr for solidification. The cellular layer was prepared by mixing of 1 × 10⁶ SKPs, 2 × 10⁵ human foreskin fibroblasts, and nerve tissues as described in DMEM with 10% FBS, 75% collagen I, 50 ng/ml heregulin (R&D Systems), and 5 μM forskolin (Sigma). Nerve tissues (DRG and sciatic nerve) were freshly isolated from adult Nf1^+/− mice, dissect into small pieces, and then added into the cellular layer mixture. The cellular layer was layered on top of acellular layer and incubated at 37°C for 1 hr for solidification. The rafts were kept submerged in regularly refreshed DMEM medium with 10% FBS. Two to five months later, tissue rafts were harvested and subjected to for histological and immunohistochemical analysis.