Cryopreservation of Human Limbal Stem Cells Ex Vivo Expanded on Amniotic Membrane

Abstract

After cornea transplantation, the donor's limbal zone is currently discarded as medical waste. However, the limbal zone is rich in limbal stem cells and can be used in therapeutic applications of limbus loss. This study aimed to increase the availability of limbal stem cells and develop the optimal conditions of cryopreservation for ex vivo expanded limbal stem cells. Pieces of the limbus were cultured on amniotic membrane (AM) to outgrow limbal stem cells as cell sheets for 3 weeks. Different formulas of cryoprotectants were tested to preserve the expanded cell sheets in liquid nitrogen. Before and after cryopreservation, expanded cell sheets were assessed for cellular characteristics by viability, histologic examination, and expression of ABCG2, vimentin, and keratin 3. Expanded cell sheets usually exhibited 3-6 stratified layers after 3-week culture on AM and expressed specific markers of ABCG2 and vimentin for limbal stem cells. The effects of cryopreservation with different cryoprotectants were analyzed by histopathology, stem cell markers, and cell viability. The results showed that the optimal formula of cryoprotectants for expanded limbal cell sheets was 60% Dulbecco modified Eagle medium, 30% fetal bovine serum, and 10% dimethyl sulfoxide. After 8-week cryopreservation in liquid nitrogen, the characteristics of limbal stem cells were maintained, and the average viability of thawed cells was 53.8% +/- 5.8%. These results showed that limbal stem cells expanded on AM could be cryopreserved and provide a promising source without delay, if banking, for patients with limbal stem cell deficiency in the future.