Translational toolkit for reproducible, cross-study profiling of human ageing hallmarks in human blood and tissue

Abstract Background Ageing is a complex, multi-dimensional process, underpinned by interacting biological hallmarks that collectively contribute to functional decline and increased susceptibility to disease. While considerable progress has been made in delineating individual ageing pathways, translation into human studies has been hindered by methodological heterogeneity and a lack of standardised, multi-system approaches. Here, we describe a validated, high-resolution toolkit for the simultaneous quantification of multiple ageing hallmarks in clinically accessible human samples, encompassing cellular senescence, immune ageing, inflammation, mitochondrial function, mTOR signalling, autophagy, genomic instability, and stem cell exhaustion. Methods Blood (25ml) was obtained from young and aged donors (26–81y). Deep immunophenotyping was performed using a novel 30-colour spectral flow cytometry panel. T-cell mTOR activation and autophagic flux were assessed by flow cytometry. Metabolic flux was measured by Seahorse. From whole blood (4 ml), muscle, and adipose tissue (AT) (obtained during elective hip arthroplasty) RNA, DNA, AT stem cells, and myoblasts were isolated. DNA copy number and senescent cell burden were assessed by q-PCR and SA-β-gal staining, respectively. Findings Utilising this toolkit, we identified pronounced age-related immune remodelling, increased senescent T-cell burden, diminished mitochondrial capacity and altered mTOR–autophagy signalling between healthy young and aged donors. Furthermore, metabolism was significantly affected by anti-coagulant and freezing sample before analysis. Interpretation This integrated platform provides a foundation for reproducible, cross-study analyses and facilitates translational investigation of interventions targeting health-span extension. Funding Wellcome Leap Dynamic Resilience program (co-funded by Temasek Trust).