Differential gene expression in Nintedanib-treated 3D skin rafts with Scleroderma (SSc) skin and healthy fibroblasts
This study examines differential gene expression in 3D skin organoids treated with Nintedanib. Overall design: Human SSc/ healthy fibroblast samples (400,000 cells/ml) were resuspended in a buffered solution of rat tail type I collagen (4 mg/ml, from BD Biosciences) and seeded in 12-well cell culture plates. The dermal collagen plugs were allowed to polymerize for 30 minutes and then supplemented with 20% Fetal Bovine Serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) overnight. 1 mL of primary human epidermal keratinocytes (600,000 cells/ml) were seeded on top of the dermal collagen plug in E medium. After 48 hours of incubation, the skin equivalents were transferred to the top of a metal grid raised above the surface of a 60-mm tissue culture dish and exposed to air to promote epidermal stratification. Approximately 2 hours prior to this, the media was changed for fresh E media supplemented with epidermal growth factor (5ng/ml). E media without epidermal growth factor was used to replace media every other day for up to 10 days. Rafts were treated with either Nintedanib (2uM) or vehicle every day. After 10 days, the rafts were harvested in RNAlater stabilization solution prior to RNA isolation.