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Abstract
Purpose
To investigate the possibility of transplantation into rabbits of collagenase-induced cultured human corneal endothelial cell plate-rabbit corneal stromal complexes.
Methods
Human corneal endothelial cells were isolated from residual corneal limbus samples and treated with collagenase to create corneal endothelial cell sheets. Cultured sheets were transplanted into the rabbit stroma after the rabbit corneal endothelial cells and Descemet's membrane were removed. Hematoxylin-and-eosin staining and immunofluorescence staining for collagen VIIIa2 (COL8A2) and zonula occludens-1 (ZO-1) were performed. The cultured human corneal endothelial cell sheet-rabbit corneal stromal complex was transplanted into rabbits. On days 3, 5, and 7, the transplanted corneas were photographed and corneal opacity was measured. One week later, the rabbits were sacrificed. Hematoxylin-and-eosin staining and immunofluorescence staining for COL8A2 were performed.
Results
The cultured cells were immunofluorescently stained for collagen VIIIa1 and ZO-1. Collagenase-treated cultured human corneal endothelial cells formed monolayers on day 7 after transplantation into the rabbit corneal stroma and immunofluorescently stained for COL8A2 and ZO-1. The cultured human corneal endothelial cell sheet-rabbit stroma complex transplanted into rabbits was transparent on days 3 and 5, but corneal opacity developed by day 7. Histologic examination revealed 3,3′-dioctadecyloxacarbocyanine perchlorate (DIO)-stained corneal endothelium (green) and hard-tissue lymphocytes had infiltrated the cultured corneal endothelial cell plate-rabbit corneal stromal complex graft group.
Figures and Tables
Figure 1
Human corneal endothelial cell (HCEC) culture and immunofluorescence staining. Cultured HCECs expressed collagen VIIIa2 (green) which is a marker of corneal endothelial cells (A), and ZO-1 (green) which is an indicator of tight junction (C). Negative staining was performed with the omission of a primary antibody (B, D). Scale bar = 50 µm.
Figure 2
Detachment and reattachment of cultured human corneal endothelial cell sheets. (A) Cultured human corneal endothelial cell sheets using collagenase were detached from the bottom of the cell dishes and floating in the media (black arrow). (B) Cultured human corneal endothelial cell sheets using collagenase were reattached onto the fibronectin and collagen-coated slides (black arrow). (C) Cultured human corneal endothelial cell sheets using collagenase were immune-stained with ZO-1 (green) after reattachments.
Figure 3
Histology of 3,3′-dioctadecyloxacarbocyanine perchlorate (DIO), hematoxylin and eosin staining and immunofluorescent staining of COL8A2 and ZO-1 of cultured human corneal endothelial cell sheets-rabbit corneal stroma complex. (A) DIO-stained cells (green) were identified on the corneal stroma. (B) Hematoxylin and eosin staining showed the mono-layer human corneal endothelial cells on the corneal stroma. (C, D) Transplanted human corneal endothelial cell sheets expressed COL8A2 (C; green) and ZO-1 (D; green) on the corneal stroma. Magnification ×200.
Figure 4
Representative pictures of in vivo transplantation of cultured human corneal endothelial cell sheets-rabbit corneal stroma complex (A) and corneal opacity grade (B). (A) Cultured human corneal endothelial cell sheets-rabbit corneal stroma complex (3 photos above) showed more transparent compared with control (3 photos below). (B) Corneal opacity grade in the experimental group was lower compared to the control group at day 1, 3 and 5. At day 7, cornea in the experimental group became more opaque than baseline. *Statistiscally significant by independent t-test.
Figure 5
Histology showed 3,3′-dioctadecyloxacarbocyanine perchlorate (DIO)-stained cells (green) on the corneal endothelium at day 7. Magnification ×200.
Figure 6
Hematoxylin and eosin staining (A), and immune fluorescence staining with collagen type VIII alpha 2 (B) at day 7. (A) Transplanted human corneal endothelial cells were on the inner surface of cornea. A few inflammatory cells were shown (black arrow), which indicates the graft rejection or graft primary failure. (B) Collagen type VIII alpha 2-expressing cells were on the inner surface of cornea. Magnification ×200.
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