Senescence in cultured trabecular meshwork cells

Background: It has been suggested that replicative senescence might be involved in the pathophysiology of age-related diseases. Aim: To study the process of senescence in trabecular meshwork (TM) cells. Methods: Porcine TM tissues were obtained and placed in primary cultures with Dulbecco's modified Eagle's medium/Ham's F-12 medium. After 2-3 weeks, migrated and proliferated TM cells were trypsinised and cultured in serial passages, and identified with fluorescein-labelled low-density lipoprotein (Dil-Ac-LDL), a marker of TM cells. Staining for senescence-related beta-galactosidase activity was performed at population doubling level (PDL) 2, 8 and 16 at pH 6. Terminal restriction fragment (TRF) length was examined by Southern blot analysis using a P-32-labelled telomere-specific sequence ( TTAGGG)(3) at each PDL. Results: Dil-Ac-LDL staining revealed that most (nearly 100%) of the cells in the culture were TM cells, which were flattened in shape and positive for senescence-related beta-galactosidase staining at PDL 16. Reduction of TRF length as a function of population doubling was also shown. Conclusions: TM cells exhibited characteristics of senescence at PDL 16 in vitro. The results demonstrated that cellular senescence may be related to the pathophysiology of primary open-angle glaucoma.