Cellular senescence, a driver of aging and age-related diseases, is a stable state found in metabolically active cells characterized by irreversible cell growth arrest and dramatic changes in metabolism, gene expression and secretome profile. Endogenous regeneration efficacy of mesenchymal stem cells (MSCs) could be attenuated due to senescence. MSCs can be modulated by not only biochemical signals but also by physical cues such as substrate topography. To provide a cell culture substrate that can prevent MSC senescence over an extended period of in vitro cultivation, here, the cell- and immunocompatible poly(ether imide) (PEI) substrate was used. Two distinct levels of roughness were created on the bottom surfaces of PEI inserts via injection molding: Low-R (similar to the thickness of attached single MSC, Rq: 3.9 ± 0.2 µm) and High-R (larger than single MSC thickness. Rq: 22.7 ± 0.8 µm). Cell expansion, lysosomal enzymatic activity, apoptosis and paracrine effects of senescent MSCs were examined by cell counting, detection of senescence-associated β-galactosidase (SA β-gal), Caspase 3/7, and CFSE labeling. MSCs showed high cell viability and similar spindle-shaped morphology on all investigated surfaces. Cells on Low-R presented the highest expansion (80000 ± 1805 cells), as compared to cells on smooth PEI and High-R. The low apoptosis level (0.08 vs 0.12 from smooth PEI) and senescence ratio (35% vs. 54% from smooth PEI) were observed in MSCs cultured on Low-R. The secretome from Low-R effectively prevents senescence and supports the proliferation of neighboring cells (1.5-fold faster) as compared to the smooth PEI secretome. In summary, the Low-R PEI provided a superior surface environment for MSCs, which promoted proliferation, inhibited apoptosis and senescence, and effectively influenced the proliferation of neighboring cells via their paracrine effect. Such microroughness can be considered as a key parameter for improving the therapeutic potential of endogenous regeneration, anti-organismal aging and anti-age-related pathologies via directly promoting cell growth and modulating paracrine effects of the senescence associated secretome.