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The increased use of ophthalmic products in recent years has led to an increased demand for in vitro and in vivo transcorneal drug absorption studies. Cell-culture models of the human cornea can avoid several of the disadvantages of widely used animal experimental models, including ethical concerns and poor standardisation. This study describes the development of a serum-free cultivated, three-dimensional human cornea model (Hemicornea, HC) for drug absorption experiments. The impact of varying cultivation conditions on the corneal barrier function was analysed and compared with excised rabbit and porcine corneas. The HC was cultivated on permeable polycarbonate filters using immortalised human keratocytes and a corneal epithelial cell line. The equivalence to native tissue was investigated through absorption studies using model substances with a wide range of molecular characteristics, including hydrophilic sodium fluorescein, lipophilic rhodamine B and fluorescein isothiocyanate (FITC)-labelled macromolecule dextran. To study the intra-laboratory repeatability and construct cultivation, the permeation studies were performed independently by different researchers. The HC exhibited a permeation barrier in the same range as excised animal corneas, high reproducibility and a lower standard deviation. Therefore, the HC could be a promising in vitro alternative to ex vivo corneal tissues in preclinical permeation studies.