Requirement for vitamin A in long-term culture of human cornea.

PURPOSE: To develop an in vitro model for the study of vitamin A functions in the cornea by determining the morphologic responses to its absence and to the addition of varying concentrations in long-term (21-day) human organ culture.

METHODS

Donor corneas were cultured for 21 days in an air-liquid environment designed to maintain normal corneal morphology in long-term culture. Corneas were graded by light microscopy and were further analyzed by transmission electron microscopy.

RESULTS

Epithelium of human corneas cultured in the absence of vitamin A for 21 days showed characteristics of vitamin A deficiency: multilayering of squamous cells, bundling of tonofilaments to form keratofibrils, increased desmosomes, and loss of microplicae on the surface cells. The cells responded to the addition of vitamin A in concentrations ranging from 5 x 10(-9) to 5 x 10(-6) M. At the lowest concentration, cell differentiation and morphology appeared normal. Cells developed secretory morphology at higher concentrations of vitamin A, with intracellular vesicles, basement membrane overproduction, and reduced cell-cell and cell-substrate attachments. Keratocytes and endothelial cells showed increased intracellular vacuoles in the presence of higher concentrations of vitamin A.

CONCLUSIONS

Cultured adult human corneas showed primary, dose-related responses to vitamin A in the absence of inflammatory, neuronal, or humoral factors. The epithelium was most sensitive to the absence of vitamin A and showed a dose-related response to additional vitamin A. All corneal cell layers showed morphologic changes at the highest concentration of vitamin A used.