Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture

Developers

Sanaz Gidfar, Farnoud Y. Milani, Behrad Y. Milani, Hossein Sagha, Ali R. Djalilian, etc.

Description of the technology

Rapamycin has previously been shown to have anti-aging effects in cells and organisms. In this technology, rapamycin was used to prolong the survival of human corneal epithelial cells in vitro. It was found that Rapamycin treatment keeps the cells in a less differentiated and less proliferative state resulting in less replicative senescence and less apoptosis.

Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay.

The results demonstrated that primary human corneal epithelial cells treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of human corneal epithelial cells prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells.

Practical application

Rapamycin treatment in accordance with this technology allows to prevent replicative senescence (Hayflick limit) in culture of primary corneal epithelial cells. It is crucial for both researches of processes of ageing (particularly, cell senescence) and practical application of cell therapies and transplantology (for example, maintenance of viability of primary corneal epithelial cells in vitro for corneal transplantation).

Laboratories

• Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago (USA)

Links

Publications

• Gidfar, S. et al. «Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture." 7 Scientific Reports, (2017): 40308.
• Milani, B. Y. et al. Rapamycin inhibits the production of myofibroblasts and reduces corneal scarring after photorefractive keratectomy. Invest Ophthalmol Vis Sci. 54(12), 7424–7430 (2013).