Epithelial adhesion complexes and organ culture of the human cornea

The effects of extended organ culture of human cornea on the structural integrity, particularly adhesion complexes, of the epithelium were determined. Human corneas were placed in organ culture using an immersion method. The structure of the cornea prior to culture (0 h) and at 7, 14, and 18 days in culture was evaluated by staining with hematoxylin/eosin, and by ultrastructural analysis that included a morphometric study of the type and number of adhesion complexes. Human corneas prepared immediately (0 h) and those in culture after 7 days showed similar structural organization and anatomical features. In contrast to 0 h specimens, the corneal epithelium at 14 days in culture exhibited signs of deterioration, with increases in cellular contraction, extracellular space, electron density of the cytoplasm, nuclear invaginations, and nucleoplasmic opacity, as well as aggregations of junctional complexes between cells. At 18 days in culture, the ocular surface epithelium was markedly reduced in thickness and consisted of no more than 2-3 cell layers; a distinct basal layer was not detected, and the morphology of the suprabasal and basal layers were similar. The basement membrane was disorganized, and anchoring complexes composed of hemidesmosomes were often absent. The number and type of the anchoring complexes associated with the basal epithelium and Bowman's membrane were comparable until 14 days of age, although the total number of hemidesmosomes per microm of epithelial plasmalemma was subnormal. After 2 weeks in culture, there were 38-72% fewer anchoring complexes and a decrease of 44% in the number of hemidesmosomes/microm of membrane from samples prepared immediately and after 7 days in culture. These results indicate that the structural integrity of human corneal epithelium in organ culture is compromised after 14 days in vitro using an immersion system of tissue culture. Thus, long-term use of cultures to define homeostasis and wound healing of the ocular surface epithelium, which necessitates normal architecture including anchoring complexes between epithelium and Bowman's membrane, may not be appropriate and requires careful monitoring both qualitatively and quantitatively at the electron microscopic level of resolution.