Human keratocytes cultured on amniotic membrane stroma preserve morphology and express keratocan

PURPOSE: To develop a new method of expanding human corneal keratocytes in serum while maintaining their characteristic morphology and keratocan expression. METHODS: Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/mL of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. On confluence, cells on AM were continuously subcultured for six passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were reseeded on AM. Cellular morphology and cell-cell networks were assessed by phase-contrast microscopy and a cell viability assay, respectively. Expression of keratocan was determined by RT-PCR and Western blot analysis. RESULTS: Trephined stroma yielded 91,600 +/- 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM, even in 10% FBS but fibroblastic on plastic. Such a difference in morphology correlated with expression of keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM, even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passages 3 and 11 did not reverse their morphology or synthesize keratocan when reseeded on plastic in 1% FBS or on AM. CONCLUSIONS: Human keratocytes maintain their characteristic morphology and keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.