Extracellular Matrix Formation in Cell Cultures

Both HCFs and HKCs were plated on transwell 6-well plates containing polycarbonate membrane inserts with 0.4 μm pores (Costar, Charlotte, NC, USA,) at a density of 10⁶ cells/mL. The protocol followed was identical for both cell types. Cells were cultured in EMEM with 10% FBS and 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (VitC, Wako Chemicals USA, Inc.; Richmond, VA, USA). The cultures were allowed to grow for 4 weeks in the presence of 0.1ng/mL TGF-β1 (T1) or TGF-β3 (T3). Cultures without any growth factors served as Controls (C). Culture medium was changed every other day for the duration of the experiment. The morphology of the cultures was examined using transmission electron microscopy (TEM). In addition, indirect-immunofluorescence was used to identify specific markers of stromal components—Smooth muscle actin (SMA), type I collagen (Col I), type III collagen (Col III), type V collagen (Col V) and fibronectin (EDA-Fn).