Development of an optimised culture medium for keratocytes in monolayer.

Unlabelled: Our objective was to formulate a medium for monolayer culture optimising both keratocyte growth and preservation of the keratocyte phenotype.

Methods: An experimental matrix selected 14 media to test, using 7 components. Selection criteria were growth rates over 5 passages and expression of the CD34 marker.

Results: Acetylcholine, insulin and vitamin C had no effect on growth and differentiation. The DMEM + Ham F12 1 : 1 based medium was selected for its initial effect on growth. At concentrations of 5 ng/ml, b-FGF improved the percentage of CD34+ cells without reducing growth rates. New-born calf serum (NCS) had a greater effect on growth than foetal calf serum (FCS). We showed three major interactions: between b-FGF and IGF-1, FCS and IGF-1 and NCS and b-FGF.

Conclusion: We selected the following medium, which provides optimal growth and preservation of the CD34+ phenotype: DMEM/HAM-F12 + 10% NCS + 5 ng/ml b-FGF + antibiotics.