Serum-free SFM corneal organ culture medium but not conventional MEM organ culture medium protects human corneal endothelial cells from apoptotic and necrotic cell death

Thekla Jaeckel (1) , Lilla Knels (1) , Monika Valtink (1) , Richard Hw Funk (2) , Katrin Engelmann (3)

1 Institute of Anatomy, TU Dresden
2 Institute of Anatomy and Center for Regenerative Therapies, TU Dresden
3 Department of Ophthalmology, Klinikum Chemnitz, and Center for Regenerative Therapies, TU Dresden

Aim: To evaluate the influence of organ culture media on corneal endothelial cell survival. Methods: The human corneal endothelial cell line HCEC-12 was cultured in five different media: HCEC growth medium F99, standard corneal organ culture medium MEM+2% FCS, MEM+5% FCS, and humanised, serum-free medium Endothelial-SFM (with and without antibiotics). Part of the cells were treated with 0.5µmol/l staurosporine and examined for signs of apoptosis by assessing mitochondrial membrane polarisation state (intravital JC-1 staining), YO-PRO-1 and propidium iodide staining, determining fragmentation of nuclei by sub-G1 DNA content, immunocytochemistry for cleaved caspase-3, cleaved caspase-8, Bax, Bcl-2, and western blotting for cleaved caspase-3 and cleaved PARP. Results: The number of apoptotic cells in untreated control cultures was significantly higher in MEM compared to F99 and SFM. Staurosporine treatment induced apoptosis in all tested cultures to varying degrees. Cells cultured in MEM showed stronger staining for cleaved caspase-3, cleaved caspase-8, Bax, Bcl-2, and cleaved PARP, increased sub-G1 DNA content, more propidium iodide and YO-PRO-1 positive cells, and more mitochondria with depolarised membranes. All parameters were significantly higher in MEM compared to F99 and SFM. SFM cultures were significantly less susceptible to cell stress. Conclusion: SFM is superior to MEM in promoting HCEC survival.

Mots clés: Cornea; Experimental &#8211 laboratory; Eye (Tissue) Banking

Thekla Jaeckel, Lilla Knels, Monika Valtink, Richard Hw Funk, Katrin Engelmann. Serum-free SFM corneal organ culture medium but not conventional MEM organ culture medium protects human corneal endothelial cells from apoptotic and necrotic cell death. British Journal of Ophthalmology, 2010, 95 (1), pp.123. ⟨10.1136/bjo.2010.183418⟩. ⟨hal-00588493⟩