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Purpose The shortage of donor corneas represents a worldwide problem, and corneal endothelial cell (CEC) therapy might be a promising alternative approach. According to the literature, different approaches have been envisaged and CECs have been either injected or grafted after being cultured onto a scaffold. Our goal would be to use donor corneas that are not suitable for transplantation, and therefore discarded (mainly elderly old donor corneas with low endothelial cell densities), to isolate and grow CECs and obtain Advanced Therapy Medicinal Products (ATMP) suitable for clinical use. We believe that endothelial cell densities (ECDs) >2000 cells/mm2, a cut-off value that eye banks normally use to provide quality tissues for transplantation to surgeons, should also be adopted as a parameter to define the quality of CECs. However, obtaining suitable CEC cultures with ECDs >2000 cells/mm2 has proven to be difficult, especially when isolated from elderly donor corneas with ECDs <2000 cells/mm2. In order to bypass such limitation, we investigated a new approach based on the injection of cell suspensions collected directly from elderly donor corneas with ECDs < 2000 cells/mm2 (and therefore not suitable for transplantation), as a new strategy for patients with Fuchs’ dystrophy and bullous keratopathy.Methods We isolated CECs from 2 elderly donors’ corneas (same donor) with ECDs <2000 cells/mm2, pooled the cells and plated the CECs onto 1 recipient donor cornea. Different protocols were compared: (1) recipient corneas with or without Descemet Membrane (DM) (+DM, i.e., endothelial cell denuded corneas; -DM, i.e., stripped corneas); (2) different culture media/conditions (standard CEC culture conditions with CEC culture medium at 37°C with 5% CO2 compared to cornea standard preservation conditions with Storagix medium at 31°C). After 15 days of culture, alizarin staining and immunofluorescence (with antibodies against ZO-1, Na+/K+ ATPase, Ncad, Lam5, Col8A2, Coll4) were performed to evaluate the ECDs and parameters, such as marker expression, polymegathism and pleomorphism of the CEC grafts obtained.Results Our results show that CEC suspensions isolated from 2 corneas of one donor can be plated on one single cornea (thus mimicking an injection protocol) and lead to a functional CEC layer with ECD >2000 cells/mm2 and standard CEC marker expression. Similar results were obtained for all the conditions investigated.Conclusions Corneal endothelial cell therapy using elderly donor corneas with ECDs <2000 cells/mm2 could be a potentially interesting strategy for patients with Fuchs’ dystrophy and bullous keratopathy. In addition, it would allow to use donor corneas that would normally be discarded. Ultimately, our strategy would help reducing the donor cornea shortage and the demand for cornea transplantation. Further investigations will have to follow, but we believe that such preliminary results would open up a new promising and challenging approach in the field of CEC ATMP.