Silk fibers assisted long-term 3D culture of human primary urinary stem cells via inhibition of senescence-associated genes: Potential use in the assessment of chronic mitochondrial toxicity

Despite being widely applied in drug development, existing in vitro 2D cell-based models are not suitable to assess chronic mitochondrial toxicity. A novel in vitro assay system mimicking in vivo microenvironment for this purpose is urgently needed. The goal of this study is to establish a 3D cell platform as a reliable, sensitive, cost-efficient, and high-throughput assay to predict drug-induced mitochondrial toxicity. We evaluated a long-term culture of human primary urine-derived stem cells (USC) seeded in 3D silk fiber matrix (3D USC-SFM) and further tested chronic mitochondrial toxicity induced by Zalcitabine (ddC, a nucleoside reverse transcriptase inhibitor) as a test drug, compared to USC grown in spheroids. The numbers of USC remain steady in 3D spheroids for 4 weeks and 3D SFM for 6 weeks. However, the majority (95%) of USC survived in 3D SFM, while cell numbers significantly declined in 3D spheroids at 6 weeks. Highly porous SFM provides large-scale numbers of cells by increasing the yield of USC 125-fold/well, which enables the carrying of sufficient cells for multiple experiments with less labor and lower cost, compared to 3D spheroids. The levels of mtDNA content and mitochondrial superoxide dismutase(2) [SOD2] as an oxidative stress biomarker and cell senescence genes (RB and P16, p21) of USC were all stably retained in 3D USC-SFM, while those were significantly increased in spheroids. mtDNA content and mitochondrial mass in both 3D culture models significantly decreased six weeks after treatment of ddC (0.2, 2, and 10 μM), compared to 0.1% DMSO control. Levels of complexes I, II, and III significantly decreased in 3D SFM-USC treated with ddC, compared to only complex I level which declined in spheroids. A dose- and time-dependent chronic MtT displayed in the 3D USC-SFM model, but not in spheroids. Thus, a long-term 3D culture model of human primary USC provides a cost-effective and sensitive approach potential for the assessment of drug-induced chronic mitochondrial toxicity.