Development of an optimised culture medium for keratocytes in monolayer.
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.


CONCLUSIONS
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.
Builles
Nicolas
N
Banque de Cornées des Hospices Civils de Lyon, 69437 Lyon, France.
Bechetoille
Nicolas
N
Justin
Virginie
V
Ducerf
Amélie
A
Auxenfans
Céline
C
Burillon
Carole
C
Sergent
Michèle
M
Damour
Odile
O
D000109:Acetylcholine / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D001205:Ascorbic Acid / Q000378:metabolism; D002417:Cattle; D049109:Cell Proliferation; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism*; D005346:Fibroblast Growth Factors / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype
D000199:Actins; D018952:Antigens, CD34; D003470:Culture Media; D007328:Insulin; D005346:Fibroblast Growth Factors; D007334:Insulin-Like Growth Factor I; D000109:Acetylcholine; D001205:Ascorbic Acid