Background <p>Keratocyte cells residing in the corneal stroma produce extracellular matrix (ECM) proteins and support transparency. These cells are pivotal for corneal regeneration and the development of in vitro tissue substitutes. However, there is limited literature on the characteristics of primary and immortalized keratocyte cells.</p> Methods and results <p>This study investigates the preservation of keratocyte phenotype in primary cells isolated from human donors and their subsequent immortalized passages. Primary human corneal keratocyte cells (hKCs) isolated from donor corneas were immortalized using SV40. The differentiated expression of corneal and limbal genes in both primary hKC (at passage 2) and immortalized hKCs (at passages 9, 12, and 15) were evaluated using RT-PCR. Additionally, cells were examined using immunocytochemistry (ICC) with corneal keratocyte markers.</p> <p>The results demonstrate the presence of myofibroblast transformation in primary hKCs, while keratocyte characteristics remain intact even in subsequent passages of immortalized cells. The highest aquaporin 1 (AQP1), aldehyde dehydrogenase-3A1 (ALDH3A1), and collagen type I (COL1) expression was observed in immortalized hKC at passage 12.</p> Conclusions <p>These findings suggest that immortalized corneal keratocytes, are a viable source for corneal tissue engineering studies, particularly up to passage 12. The isolation, characterization, and immortalization of corneal keratocytes are imperative for further research and applications. Knowing the characteristics of keratocyte cells used in tissue engineering and therapeutic studies will significantly impact the outcomes.</p>