Construction of a Complete Rabbit Cornea Substitute

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(Investigative Ophthalmology and Visual Science. 2006;47:3311-3317.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI: 10.1167/iovs.05-1647
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Construction of a Complete Rabbit Cornea Substitute Using a Fibrin-Agarose Scaffold[/B]

Miguel Alaminos,1,2 María Del Carmen Sánchez-Quevedo,1 José Ignacio Muñoz-Ávila,3 Daniel Serrano,3 Santiago Medialdea,4 Ignacio Carreras,5 and Antonio Campos1

1From the Departments of Histology and 5Surgery, University of Granada, Granada, Spain; 2Fundación Hospital Clínico, Granada, Spain; the 3Division of Ophthalmology, University Hospital San Cecilio, Granada, Spain; and the 4Division of Ophthalmology, University Hospital Virgen de las Nieves, Granada, Spain.

PURPOSE. To construct a full-thickness biological substitute of the rabbit cornea by tissue engineering.

METHODS. Ten rabbit corneas were surgically excised, and the three main cell types of the cornea (epithelial, stromal, and endothelial cells) were cultured. Genetic profiling of the cultured cells was performed by RT-PCR for the genes COL8 and KRT12. To develop an organotypic rabbit cornea equivalent, we used a sequential culture technique on porous culture inserts. First, endothelial cells were seeded on the base of the inserts. Then, a stroma substitute made of cultured keratocytes entrapped in a gel of human fibrin and 0.1% agarose was developed. Finally, cultured corneal epithelial cells were grown on the surface of the scaffold. Stratification of the epithelial cell layer was promoted by using an air–liquid culture technique. Corneal substitutes were analyzed by light and electron microscopy.

RESULTS. All three types of corneal cells were efficiently cultured in the laboratory, expanded, and used to construct a full-thickness cornea substitute. Gene expression analyses confirmed that cultured endothelial cells expressed the COL8 gene, whereas epithelial cells expressed KRT12. Microscopic evaluation of the cornea substitutes demonstrated that epithelial cells tended to form a normal stratified layer and that stromal keratocytes proliferated rapidly in the stromal substitute. The endothelial monolayer exhibited a pattern similar to a normal corneal endothelium.

CONCLUSIONS. These findings suggest that development of a full-thickness rabbit cornea model is possible in the laboratory and may open new avenues for research.

http://www.iovs.org/cgi/content/abstract/47/8/3311