Michael R Kozlowski1. 1. Arizona College of Optometry, Midwestern University , Glendale, AZ , USA.
Abstract
PURPOSE: The present report examines several subcultures of a single sample of ARPE-19 cells to determine their status with respect to cell mortality. If a transformation from mortal to immortal has occurred in these cells, it may impact their characteristics and, thereby, their utility for modeling natural retinal pigment epithelial (RPE) cells. METHODS: Five separate subcultures of ARPE-19 cells were grown as recommended by the supplier. During the course of culture, they were periodically monitored for signs of mortality including erosion of telomeres, increased senescence-associated beta-galactosidase (SABG) staining, altered morphology and reduced viability with an increased population doubling level (PDL). There were also observed for signs of immortality including continuous growth to very high population doubling levels and maintenance of short telomere lengths. RESULTS: Each of the subcultures showed both mortal and immortal characteristics. Telomere erosion, increased SABG staining, changes in cell morphology and a modest drop in cell viability took place within a range of population doublings (59-77) in which cell senescence would be expected to occur. The cultures, however, continued to proliferate even after signs of senescence had appeared, with one subculture propagating to 257 population doublings. In addition, little further telomere erosion occurred at high PDL. CONCLUSION: These results suggest that the ARPE-19 subcultures contained both mortal and immortal cells. Since no transformation event was witnessed during the study, it appears likely that both types of cells were present in the original sample. Based on the proportion of cells demonstrating senescence-related changes, the mortal cells were estimated to comprise approximately 27% of the total culture. Because of the differences that can exist between normal and immortalized cells, and given the large proportion of ARPE-19 cells that are immortalized, discretion should be exercised when using ARPE-19 cells to model native RPE cells for the study of retinal diseases such as AMD.
PURPOSE: The present report examines several subcultures of a single sample of ARPE-19 cells to determine their status with respect to cell mortality. If a transformation from mortal to immortal has occurred in these cells, it may impact their characteristics and, thereby, their utility for modeling natural retinal pigment epithelial (RPE) cells. METHODS: Five separate subcultures of ARPE-19 cells were grown as recommended by the supplier. During the course of culture, they were periodically monitored for signs of mortality including erosion of telomeres, increased senescence-associated beta-galactosidase (SABG) staining, altered morphology and reduced viability with an increased population doubling level (PDL). There were also observed for signs of immortality including continuous growth to very high population doubling levels and maintenance of short telomere lengths. RESULTS: Each of the subcultures showed both mortal and immortal characteristics. Telomere erosion, increased SABG staining, changes in cell morphology and a modest drop in cell viability took place within a range of population doublings (59-77) in which cell senescence would be expected to occur. The cultures, however, continued to proliferate even after signs of senescence had appeared, with one subculture propagating to 257 population doublings. In addition, little further telomere erosion occurred at high PDL. CONCLUSION: These results suggest that the ARPE-19 subcultures contained both mortal and immortal cells. Since no transformation event was witnessed during the study, it appears likely that both types of cells were present in the original sample. Based on the proportion of cells demonstrating senescence-related changes, the mortal cells were estimated to comprise approximately 27% of the total culture. Because of the differences that can exist between normal and immortalized cells, and given the large proportion of ARPE-19 cells that are immortalized, discretion should be exercised when using ARPE-19 cells to model native RPE cells for the study of retinal diseases such as AMD.
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