Background: One of the major limitations in studying the aging process is the isolation of naturally-occurring senescent cells from younger animals. Consequently, senescent cells are generated by treatment with radiation, chemical agents, or derived from genetic models of accelerated aging. However, none of these approaches fully recapitulates physiological aging at the cellular level. Senescent cells have a lower nucleus/cytoplasm ratio, which yields a lower specific density compared to young cells. It is possible to make use of this characteristic to separate senescent and young cells in mixed primary or other cultures.
Purpose: To establish a method to enrich senescent cells from physiologically aging animals at any age to provide an enriched cell source to study mechanism(s) of cellular aging.
Method: We established a new method of enriching physiologically aged bone marrow stromal cells(BMSCs) using a Percoll density gradient centrifugation technique. We isolated BMSCs from 2 month old mice and confirmed that they were over 80% Scal-1+ CD29+ CD11b- CD45- CD105-. As proof-of-principle, cells were aged in vitro (and senescence confirmed by low BrdU incorporation) and underwent Percoll density gradient centrifugation. Compared to cells that were not aged in vitro cells isolated from the lower density gradient were over 80% enriched for senescent cells as characterized by SA-β-Gal staining. Cells present in the higher density gradient layers of the gradient were enriched for younger cells. Isolated cells from each layer were compared by ability to differentiate into osteoblasts, adipocytes, and chondrocytes.
Conclusion: This density-based method of gradient centrifugation can separate young and senescent BMSCs.
Significance: This enrichment method can be ulitized to isolate and study mechanisms of physiologic cell aging and may have implications for the use of BMSC in clinical transplantation applications.