Serum-free corneal organ culture medium (SFM) but not conventional minimal essential organ culture medium (MEM) protects human corneal endothelial cells from apoptotic and necrotic cell death
| Autor: | Lilla Knels, Katrin Engelmann, Richard Funk, Monika Valtink, Thekla Jäckel |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Programmed cell death
Cell Survival Organ Preservation Solutions Apoptosis Biology Organ culture Culture Media Serum-Free Cell Line 03 medical and health sciences Cellular and Molecular Neuroscience chemistry.chemical_compound Organ Culture Techniques 0302 clinical medicine medicine Animals Humans Staurosporine Propidium iodide Fragmentation (cell biology) Cells Cultured 030304 developmental biology 0303 health sciences Endothelium Corneal Flow Cytometry Immunohistochemistry Molecular biology Sensory Systems Staining Endothelial stem cell Ophthalmology chemistry 030221 ophthalmology & optometry sense organs medicine.drug |
| Zdroj: | British Journal of Ophthalmology. 95:123-130 |
| ISSN: | 0007-1161 |
| Popis: | 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: human corneal endothelial cell (HCEC) growth medium (F99 HCEC ), standard minimal essential corneal organ culture medium (MEM)+2% fetal calf serum (FCS), MEM+5% FCS, and humanised, endothelial serum-free medium (SFM) (with and without antibiotics). A portion of the cells was treated with 0.5 μmol/l staurosporine and examined for signs of apoptosis by assessing mitochondrial membrane polarisation state (intravital JC-1 staining), by YO-PRO-1 and propidium iodide staining, by determining fragmentation of nuclei by sub-G1 DNA content, by immunocytochemistry for cleaved caspase-3, cleaved caspase-8, Bcl2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2), and by western blotting for cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Results The number of apoptotic cells in untreated control cultures was significantly higher in MEM compared with F99 HCEC 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 with F99 HCEC and SFM. SFM cultures were significantly less susceptible to cell stress. Conclusion SFM is superior to MEM in promoting HCEC survival. |
| Databáze: | OpenAIRE |
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