In the cornea, the corneal stroma is the middle layer which represents 90% of its total thickness and has an important role in its refractive behavior. The keratocytes are specific stromal cells especially responsible for the transparency maintenance of the cornea. Faced with an injury, they may lose their differentiated phenotype, transforming into dedifferentiated repair phenotypes (fibroblasts and myofibroblasts) which can produce a fibrotic scarring, causing loss of transparency of the cornea. Tissue engineering aims at developing substitutes to restore the function of a specific tissue that is damaged and the restoration of the cornea. The use of corneal substitutes requires the determination of optimal culture conditions for maintaining the differentiated phenotype of keratocytes. Recent studies reviewed in this paper showed that various factors affecting the in vitro maintenance of differentiated phenotype of keratocytes, including: use of serum, calcium concentration, several growth factors (i.e., EGF, FGF-2, insulin,