Induction of telomere shortening and replicative senescence by cryopreservation

Cryopreservation can alter cellular function under certain conditions. In t his report, we demonstrate the induction of cellular senescence after cells have been cryopreserved using a standard protocol. A retinal pigment epith elial cell line frozen at a specific freezing rate and subsequently thawed showed severely impaired proliferation compared to cells that were not cryo preserved. The induction of senescence was suggested by senescent associate d beta -galactosidase activity and diminished bromo-deoxyuridine incorporat ion. A remarkable increase of single-strand DNA breaks in terminal restrict ion fragment (TRF) were found in cryopreserved cells immediately after thaw ing. The rate of mean TRF length shortening was accelerated after cryoprese rvation. Given this evidence, we hypothesize that cryopreservation may caus e telomere shortening and cellular senescence under certain freezing condit ions. (C) 2001 Academic Press.