Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

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Stabilized collagen-glycosaminoglycan scaffolds for tissue engineered human corneas were characterized. Hydrated matrices were constructed by blending type I collagen with chondroitin sulphates (CS), with glutaraldehyde crosslinking. A corneal keratocyte cell line was added to the scaffolds with or without corneal epithelial and endothelial cells. Constructs were grown with or without ascorbic acid. Wound-healing was evaluated in chemical-treated constructs. Native, noncrosslinked gels were soft with limited longevity. Crosslinking strengthened the matrix yet permitted cell growth. CS addition increased transparency. Keratocytes grown within the matrix had higher frequencies of K+ channel expression than keratocytes grown on plastic. Ascorbic acid increased uncrosslinked matrix degradation in the presence of keratocytes, while it enhanced keratocyte growth and endogenous collagen synthesis in crosslinked matrices. Wounded constructs showed recovery from exposure to chemical irritants. In conclusion, this study demonstrates that our engineered, stabilized matrix is well-suited to function as an in vitro corneal stroma.