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Serum eye drops are used for the treatment of ocular surface disease (eg, Sicca syndrome). The objective of this experimental study was to investigate whether they maintain their wound-healing potency after a prolonged storage of 6 months at −20°C and to find a parameter that can serve as a quality and stability indicator.After obtaining whole blood from 10 volunteers and preparing 100% (AS100), 50% (AS50), and 20% (AS20) serum eye drops, epitheliotrophic factors including EGF, fibronectin, vitamins A and E, albumin, and immunoglobulin A were quantified before and after storage for 7 days at 6°C or 3 and 6 months at −20°C. Human corneal epithelial (HCE) cell lines were used to investigate proliferation, migration, and overall wound healing potency of the cells in response to different serum preparations. The proliferation, migration, and wound healing of HCE cells were measured after incubation with different serum eye drop concentrations and after different storage conditions.The concentration of epidermal growth factor, fibronectin, vitamins A and E, immunoglobulin A, and albumin showed no significant reduction over the test period. Proliferation, migration, and wound healing of HCE cells was significantly better after incubation with undiluted serum in comparison with diluted serum. No significant loss of cytokine concentration, wound healing, and proliferation effect in HCE culture of AS100, AS50, and AS20 could be detected over the 6 months of storage.The concentration of a spectrum of cytokines involved in corneal epithelial wound healing and the epitheliothrophic effect of serum are not significantly changed after a prolonged storage of 6 months at −20°C. Hence, it seems justifiable to provide patients with appropriate freezer capacity with a 6-month supply of autologous serum eye drops. Albumin—which is known to be relevant for ocular surface health—could serve as a cost-effective parameter for stability controls.