Human corneal endothelial cells (HCEnCs) are responsible for maintaining the transparency of the cornea. Damaged or diseased HCEnCs may cause blindness. Replacement of the diseased cells with a healthy donor endothelium is the only currently available treatment. Tissue-engineering can serve as an alternative to conventional donor corneal transplantation. Due to the global shortage of donor corneas, a wide interest in the development of cultured graft substitutes and artificial corneas has increased. Availability of the old donor corneas is higher especially for research. Although it can be proposed as a valuable source for cell culture, its less proliferative capability emerges a challenge for the researchers. This article describes the use of hyaluronic acid (HA) in combination with Rho-kinase inhibitor (ROCK) Y-27632 for the cultivation of HCEnCs from older donor corneas (age > 60 years). Four conditions including and excluding HA + ROCK and its effect on early attachment rates and proliferation was studied on forty-eight corneas. It was observed that HCEnCs reach confluence within 10-15 days when cultured with HA + ROCK. This approach improves the efficiency of cell adhesion due to force attachment. HCEnCs from old donor corneas can be cultured using this method which may further lead to cell-based therapy for treating corneal endothelial dysfunction.