Stem cell exhaustion plays a major role in the ageing of different tissues. Similarly, in vitro cell ageing during expansion prior to their use in regenerative medicine can severely compromise stem cell quality through progressive declines in differentiation and growth capacity. We utilized non-destructive multispectral assessment of native cell autofluorescence to investigate the metabolic mechanisms of in vitro mesenchymal stem cell (MSC) ageing in human bone marrow MSCs over serial passages (P2–P10). The spectral signals for NAD(P)H, flavins and protein-bound NAD(P)H were successfully isolated using Robust Dependent Component Analysis (RoDECA). NAD(P)H decreased over the course of hMSC ageing in absolute terms as well as relative to flavins (optical redox ratio). Relative changes in other fluorophore levels (flavins, protein-bound NAD(P)H) suggested that this reduction was due to nicotinamide adenine dinucleotide depletion rather than a metabolic shift from glycolysis to oxidative phosphorylation. Using multispectral features, which are determined without cell fixation or fluorescent labelling, we developed and externally validated a reliable, linear model which could accurately categorize the age of culture-expanded hMSCs. The largest shift in spectral characteristics occurs early in hMSC ageing. These findings demonstrate the feasibility of applying multispectral technology for the non-invasive monitoring of MSC health in vitro.

干细胞耗竭在不同组织的衰老中起着重要作用。同样，体外细胞在用于再生医学之前的扩增过程中的老化会通过分化和生长能力的逐渐下降而严重损害干细胞的质量。我们利用天然细胞自发荧光的非破坏性多光谱评估来研究体外间充质干细胞（MSC）连续传代（P2-P10）中人骨髓 MSC 老化的代谢机制。使用稳健相关成分分析 (RoDECA) 成功分离出 NAD(P)H、黄素和蛋白质结合的 NAD(P)H 的光谱信号。在 hMSC 老化过程中，NAD(P)H 的绝对值以及相对于黄素的值（光学氧化还原比）均有所下降。其他荧光团水平（黄素、蛋白质结合的 NAD(P)H）的相对变化表明，这种减少是由于烟酰胺腺嘌呤二核苷酸的消耗，而不是从糖酵解到氧化磷酸化的代谢转变。利用无需细胞固定或荧光标记即可确定的多光谱特征，我们开发并外部验证了可靠的线性模型，该模型可以准确地对培养扩增的 hMSC 的年龄进行分类。光谱特征的最大变化发生在 hMSC 老化的早期。这些发现证明了应用多光谱技术对 MSC 体外健康状况进行无创监测的可行性。