These conditions were previously described as most appropriate for keratocyte growth. By 48 hours the plated cells had attached and begun to show a dendritic morphology. By 2 weeks, the primary DN cells continued to grow in serum-free DMEM: F12/ITS and maintain their keratocyte morphology, while KC keratocytes that were morphologically similar to DN keratocytes early on, at 6 days after isolation, became rounded and began to deteriorate by 12 days. Thus, freshly isolated KC cells showed poor survival in serum-free medium, mimicking metabolic and growth impairment features of in situ keratocytes from KC corneas. Here we developed methods to study stromal cells from individual DN and KC cornea button halves. We show that single cornea-half derived stromal cells could be expanded as fibroblasts and stored at low passage numbers. In serum-free media containing ITS and phosphoascorbic acid, the fibroblasts assumed a dendritic morphology typically seen in keratocytes. We investigated TGF b signal transductions in serum-starved KC and DN fibroblasts, as multiple lines of evidence supported a role for this signaling network in keratoconus. The TGFb1 and 2 ligands are KRX-0401 present in their inactive form in the corneal epithelium under homeostatic conditions, while in their active cleaved forms they are present in the epithelium and to a lesser extent in the stroma during injury and infections. Earlier studies have shown that keratocytes respond to TGF b1 stimulus, however these were conducted in the context of myofibroblastic changes, ECM production and fibrosis. Serum-starved fibroblasts exposed to TGF b1 showed increased phosphorylation of SMAD1/5/8, and this phosphorylation was significantly higher in the KC cells. This less common route of TGFb1 signal transduction reported before in vascular endothelial cells may be operative in the corneal stroma. Moreover, this appeared to be increased in keratoconus. The canonical signaling via phosphorylation of SMAD2/3 was Carfilzomib 868540-17-4 constitutively high in DN and KC serum-starved fibroblasts with the KC cells showing higher levels that did not reach statistical significance.