To investigate the characteristics and the results of long-term follow-up of limbal epithelial cells cultivated in vivo on amniotic membranes (LIVAM) in corneal limbal deficiency.

Twenty-two eyes of twenty-two patients diagnosed with corneal limbal deficiency underwent transplantation of in vivo cultivated corneal limbal epithelial cells on the amniotic membrane. Biopsy and immunohistochemical staining (AE5, MUC5AC) of the amniotic membrane cultivated for one week were performed to verify that the cultivated epithelial cells on the amniotic membrane were corneal epithelial cells. Impression cytology was performed to evaluate the characteristics of the transplanted corneal limbal epithelial cells at postoperative 1 week, 3 months, 6 months, and 1 year.

Successful epithelial growth was observed on the amniotic membrane at one week. The epithelial cells were confirmed to be corneal epithelial cells by immunohistochemical staining. Transplanted in vivo cultivated corneal epithelial cells were confirmed to have corneal specificity by impression cytology and immunohistochemical staining at postoperative 1 week, 3 months, 6 months, and 1 year.

In vivo cultured corneal epithelial cells showed morphological and immunohistochemical findings similar to those of normal corneal epithelial cells. Transplanted in vivo cultivated corneal epithelial cells were maintained and showed the characteristics of corneal epithelial cells. Transplantation of in vivo cultivated corneal limbal epithelial cells can be performed to reconstruct the corneal limbus in treating corneal limbal deficiency.