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Ji-Eun Lee, Seung Uk Lee, Jong Soo Lee; Effect of Serum Clot Activator on Keratocytes. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5239.
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To evaluate the effect of serum clot activator which could be used for making autologous serum eye drop on human keratocytes.
Cultured human corneal keratocytes were exposed to 10, 20, and 30 % SiO2 for 24, 48, and 72 hours, MTT-based calorimetric assay was performed to determine the survival rate of keratocytes and lactate dehydrogenase (LDH) leakage assay to assess the cytotoxicity. Apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy.
The survival rate of human keratocytes and cytotoxicity showed the concentration and time dependent response and significant response was found after treating with 30% SiO2 for 72 hours. Apoptosis developed in flow cytometry and apoptotic cells were demonstrated in fluorescent micrograph after treating with 30% SiO2 for 48 hours but cells were non viable after 72 hours. Human keratocytes were more detached from the bottom of the dish and damaged cells have degenerative changes like microvilli disappearance, vacuoles formation and chromatin of the nuclear remnant condensed along the nuclear periphery after treating with 30% SiO2 for 72 hours.
SiO2, the serum clot activator, showed the toxicity on human keratocytes at the concentration of 30% after 72 hours treament. So blood should be extracted with tubes without clot activator during making the autologous serum eye drop for preventing the possible cytotoxicity on cornea.
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