Transplantation of tissue-engineered human corneal endothelium in cat models

Abstract

Purpose: To evaluate the performance of reconstructed tissue-engineered human corneal endothelium (TE-HCE) by corneal transplantation in cat models.

Methods: TE-HCE reconstruction was performed by culturing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled monoclonal HCE cells on denuded amniotic membranes (dAMs) in 20% fetal bovine serum-containing Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 (1:1) medium and 5% CO(2) at 37 ° C on a 24-well culture plate. The reconstructed TE-HCE was transplanted into cat corneas via lamellar keratoplasty with all of the endothelium and part of Descemet's membrane stripped. Postsurgical corneas were monitored daily with their histological properties examined during a period of 104 days after transplantation.

Results: The reconstructed TE-HCE at a density of 3,413.33 ± 111.23 cells/mm(2) in average established intense cell-cell and cell-dAM junctions. After lamellar keratoplasty surgery, no obvious edema was found in TE-HCE-transplanted cat corneas, which were transparent throughout the monitoring period. In contrast, intense corneal edema developed in dAM-transplanted cat corneas, which were turbid. The corneal thickness gradually decreased to 751.33 ± 11.37 μm on day 104 after TE-HCE transplantation, while that of dAM eye was over 1,000 μm in thickness during the monitoring period. A monolayer of endothelium consisting of TE-HCE-originated cells at a density of 2,573.33 ± 0.59 cells/mm(2) attached tightly to the surface of remnant Descemet's membrane over 104 days; this was similar to the normal eye control in cell density.

Conclusions: The reconstructed TE-HCE was able to function as a corneal endothelium equivalent and restore corneal function in cat models.

Figure 1

The tissue-engineered human corneal endothelium reconstructed from monoclonal human corneal endothelium (HCE) cells and denuded amniotic membrane (dAM) at day 4. The morphology and structure of tissue-engineered (TE)-HCE cells are shown in 1% alizarin red staining A. Frozen sections were stained with hematoxylin and eosin (H&E), B and visualized using scanning electron microscopy (SEM) C and transmission electron microscopy (TEM) D. Scale bars, A 20 μm; B 100 μm; C 10 μm; D 2 μm.

Figure 2

Slit-lamp biomicroscopic photographs of corneas from transplanted cats. The transparency and edema status of cat corneas in the tissue-engineered human corneal endothelium (TE-HCE) group A and denuded amniotic membrane (dAM) group B at 18, 58, and 104 days after surgery, respectively.

Figure 3

Time-course of average central corneal thickness A and the eye pressure B of transplanted cats.

Figure 4

Corneal endothelia from transplanted cats. Fluorescent photographs of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled monoclonal human corneal endothelium (HCE) cells in tissue-engineered (TE)-HCE eye A and denuded amniotic membrane (dAM) eye B alizarin red staining photographs of the monoclonal HCE cells in TE-HCE eye C and normal control eye D are shown. In the TE-HCE eye A, a cell-visible high magnification view of the cornea is shown in the box with a dashed line. Scale bars A, B 100 μm; C, D 50 μm.

Figure 5

Histological structures of corneas from transplanted cats. Representative corneal photomicrographs of hematoxylin and eosin (H&E) staining in tissue-engineered human corneal endothelium (TE-HCE) eye A and normal control eye B SEM in TE-HCE eye C and normal control eye D are shown. Scale bars A, B 100 μm; C, D 10 μm.

Figure 6

Representative transmission electron microscopy (TEM) photographs of corneas in tissue-engineered human corneal endothelium (TE-HCE) eye (A, D) normal control eye B, E and denuded amniotic membrane (dAM) eye C from transplanted cats. DM represents Descemet’s membrane. White arrow pointed out the intercellular tight junctions, white arrow heads pointed out the anchoring junctions such as adherens junctions and desmosomes, black arrow pointed out the gap junctions, while black arrow heads pointed out the mitochondria. Scale bars A, B, C 2 μm; D, E 200 nm.