In vitro model systems offered: Evercyte ´s senolytic efficacy and toxicity assays are based on human cells from different tissues and organs (e.g. skin fibroblasts, lung fibroblasts, umbilical vein endothelial cells, microvascular endothelial cells, renal proximal tubular epithelial cells, mesenchymal stem cells). Induction of senescence:
besides growing cells until they enter replicative senescence, protocols have been established to induce stress-induced premature senescence (SIPS) using doxorubicin treatment; as control, actively proliferating cells as well as quiescent cells (growth to confluence / contact inhibited) can be cultivated in parallel.Characterization of the senescent phenotype:
a series of tests have been established for monitoring the cellular phenotype such as monitoring the cell morphology, testing for SA-ß-galactosidase activity, analysis of expression of typical senescence markers (e.g. p16, p21), analysis of cell cycle progression by testing BrdU incorporation, analysis of changes in the senescence associated secretory phenotype (SASP) by measuring secretion of e.g. Il6, Il8, MCP-1 in cell culture supernatants or miRNA profiling.
Induction of senescence in human normal cells
characterization of the senescent phenotype
Normal human cells are treated with doxorubicin following established protocols to induce stress induced premature senescence (SIPS). Thereafter, the senescent phenotype is confirmed by transcriptomic analysis. As shown in above figure, a significant increase in p21 in SIPS compared to quiescent cells (QUI) is observed in all cell types analysed.
HDF: human dermal fibroblasts, ASC: adipose-derived mesenchymal stem cells, HDMVEC: human dermal microvascular endothelial cells, HUVEC: human umbilical vein endothelial cells, RPTEC: renal proximal tubular epithelial cells.
Data have been generated in cooperation with TAmiRNA.
Normal human lung fibroblasts are treated with doxorubicin following established protocols to induce stress induced premature senescence (SIPS). Whereas normal, early passage, subconfluent cells are characterized by the typical spindle-shaped mesenchymal morphology, doxorubicin treated cells aquire the typical, flattened, multi-nucleated morphology of senescent cells.
Testing senolytic activity of compounds
measurement of cellular viability
Human quiescent and senescent (SIPS) skin fibroblasts are treated with compounds and cellular viability is measured after 3 days. This allows us to identify compounds with a potential senolytic (blue) activity against senescent cells as well as cytotoxic activity (gray) against growth arrested control cells.