Transplantation of cultured bovine corneal endothelial cells to rabbit cornea: clinical implications for human studies.

Abstract

Rabbit corneas denuded of their endothelium were coated with bovine corneal endothelial cells (from steers) previously maintained in tissue culture for short (20 generations) or prolonged (200 generations) periods. When grafted back into female rabbits, the corneal buttons remained clear and showed no edema. In contrast, denuded corneas coated with bovine keratocytes and grafted into rabbits became opaque and edematous within 7 da s and remained so thereafter. Bovine corneal endothelial cells of the grafted corneas, which had remained clear for over 100 days, proliferated actively when put back into tissue culture. The corneal endothelial cells of the graft had a chromosome number of 2n = 60. The sex chromosomes were characteristic of the male (XY). The chromosome number of the endothelium of the recipient rabbit was 2n = 44 with sex chromosomes characteristic of the female (XX). Results of the karyotype analysis show that there was no invasion of the corneal button by the recipient endothelium and, conversely, no invasion of the recipient endothelium by the endothelium on the corneal button. These results demonstrate that cultured corneal endothelial cells remain functional in vitro and can replace a damaged or nonfunctional endothelium in vivo. The cornea owes its transparency to its uniform structure, avascularity, and deturgescence. The relative dehydration of the corneal tissue is determined primarily by the active transport pump of the corneal endothelium. The ability of most corneal endothelia to repair themselves in vivo is severely limited by their lack of proliferative capacity (1, 2). Although small endothelial wounds can be repaired through the process of endomitosis and cell enlargement (3) (5). The cells were maintained on gelatinized dishes in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 5% dextran, 50 ,ug of gentamycin per ml, and 2.5 .g of Fungizone per ml. Fibroblast growth factor (100 ng/ml) was added every other day (5, 6). The cultures were passaged every week with a split ratio of 1:64. The morphological and biochemical characteristics of these cultures have been described (5-10). Cultures of bovine corneal keratocytes were established as described (7).Coating of Denuded Descemet's Membrane of Rabbit Cornea with Bovine Corneal Endothelial Cells or Keratocytes. Female Dutch rabbits were used throughout this study. The procedure is summarized in Fig. 1. After being anesthetized, the animal was sacrificed and the eye was enucleated. The cornea and a rim of sclera were then excised. The cornea was placed in a petri dish so that the endothelial layer faced upward. A cotton swab was used to sweep the surface of the cornea (6). [Control studies with an alizarin red staining technique have shown that the entire endothelial cell layer is removed and that no damage is done to Descemet's membrane (6,9).] A corneal button was cut with an 1 -mm trephine and placed in a Teflon container fashioned to fit the contour of the cornea. Concurrently with the dissection and pr...

Human Corneal Endothelial Cell Expansion for Corneal Endothelium Transplantation: An Overview

“…The idea of using cultured HCECs as an alternative to full-thickness keratoplasty in the replacement of defective corneal endothelial layer was conceptualized over 3 decades ago (90,91). Potentially, the use of such tissue-engineered corneal endothelium substitutes can possibly alleviate the shortage of donor human corneal tissue faced globally.…”

Properties of Corneas Reconstructed with Cultured Human Corneal Endothelial Cells and Human Corneal Stroma

“…[6][7][8][9] We have also succeeded in primary culture and serial passaging of HCEC lines from a wide range of donor tissues. 10 Moreover, several groups have demonstrated the capacity of endothelial cells to survive and function properly when transplanted onto a nonnative cornea in species possessing 1,2,4,11 or lacking 3 regenerative capacity of their native corneal endothelium. Furthermore, recent attempts to transplant HCEC to monkey 12,13 and human recipients 14,15 have been successful.…”

“…Despite these promising achievements, much work remains in the characterization of the ex vivo properties of tissue-engineered corneas using cultured HCEC before cultured HCEC transplantation will become a feasible alternative to cornea transplantation. While several previous studies [1][2][3][4][11][12][13][14][15] have investigated the validity of tissue-engineered corneas by transplanting them to animals, few studies have examined the ex vivo function of tissue-engineered corneas. Although in vivo investigation of tissue-engineered corneas is essential prior to clinical application, quantitative analysis of the tissue-engineered corneas is difficult to perform in vivo.…”

“…[1][2][3][4] Several groups have developed techniques for primary culture and serial passaging of HCEC from embryos 5 or donor corneas of all ages. [6][7][8][9] We have also succeeded in primary culture and serial passaging of HCEC lines from a wide range of donor tissues.…”

Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation

“…Human amniotic membranes have been used as a carrier for cultured CECs [40], but because of its translucent nature, it is not highly recommended for clinical applications. Gospodarowicz et al [41][42][43] tried to combine two species by seeding bovine CECs onto rabbit corneas denuded of their endothelium. It was reported that when corneas were transplanted back into rabbits, the corneal buttons showed full transparency without any edema [41][42][43].…”