However, when we used this approach on freshly isolated stromal cells from keratoconus corneas, cell death was unacceptably high at the early stages and ultimately led to poor total cell yield. This is consistent with the idea that the keratocytes in keratoconus have some functional, metabolic deficiencies and a significant role in the thinning degenerative corneal phenotype of this disease. LY294002 Therefore, the growth potentials of patient and donor cells were further investigated using serumstarved fibroblasts that resemble keratocytes in their dendritic morphology and keratocan expression. We tested the effects of insulin and TGFb1 on cell proliferation in serum-starved fibroblasts in low and high glucose DMEM or DMEM: F12 with intermediate glucose to recreate normoglycemic and hyperglycemic conditions. We found that TGF b1 restricted or suppressed keratocyte growth in DN and KC keratocytes, and this growth suppression was counteracted by insulin in ITS. The negative regulation of cell cycle progression by TGF b is well documented in epithelial, hematopoietic and neural cells. Studies on human corneal fibroblasts have also shown suppression by TGF b and promotion of growth by insulin. Hyperglycemic conditions have been reported to induce oxidative stress in multiple cell types ; combined with serum starvation it may be detrimental to serum-starved KC patient fibroblasts, in particular. Indeed, our cell proliferation assay showed loss of cells in high glucose DMEM, while low glucose medium seemed to alleviate this effect, and significantly so in patient serum-starved fibroblasts. Moreover, serum-starved fibroblasts from donor and patient corneas could be cultured long term in low glucose medium, where they synthesized keratocan and elaborated an ECM. Insummary, this study shows that stromal cells canbe cultured and banked from individual donor and patient Trichostatin A cornea halves and reverted to a keratocyte-like morphology for functional analyses. A novel TGFb- pSMAD1/5/8 route was identified in the cornea.Corneal fibroblasts and serum-starved fibroblasts from keratoconus patients showed differential responses to insulin and TGF b1 that will be further investigated to elucidate disease-specific molecular changes.