3D Culture Systems Of Urine-Derived Stem Cell For NTRI-Induced Mitotoxicity Assessment

总结 抗逆转录病毒疗法（ART）大大降低了艾滋病毒相关的发病率和死亡率。然而，治疗 艾滋病毒感染者的预期寿命 随着抗逆转录病毒治疗的长期安全性越来越受到关注。因此， 抗逆转录病毒疗法的长期安全性。2D培养中的人肝细胞系大多数是 用于评估ART诱导的短期线粒体毒性（MtT）。然而，这些 不能用于检测ART诱导的慢性MtT，因为它们不能存活超过 在毒性测试期间。此外，抗病毒药物毒性筛查往往需要昂贵的初级 例如2D培养系统中的外周血单核细胞。超过90%的药物通过 2D培养临床前研究未能满足后续研究所需的预期疗效或安全范围。 审判显然，2D培养导致MtT筛选试验在以下方面的预测能力较差： .虽然长期毒性试验通常在动物模型中进行，但在动物模型中观察到的MtT的高比率是不可能的。 早期的临床试验表明，动物毒理学研究可能不适合预测抗病毒药物的MtT， 供人类使用。显然，没有足够的2D细胞培养或动物模型用于晚期MtT 用于临床前药物开发。因此，迫切需要从毒理学角度 和临床预测性体外测定以评估化合物的晚期MtT潜力。我们是 为了证明干/祖细胞存在于人尿中，即，尿源性干细胞（USC）。这些 可以通过简单、非侵入性和低成本的程序获得。USC表达端粒酶活性 具有强大的增殖潜力。我们最近透露，南加州大学的3D文化提供了一个长期的， 用于细胞生长和增殖的微生物技术，模拟体内条件，其可能具有 提高临床前抗病毒药物研究的预测结果。因此，在本发明中， 是开发用于ART诱导的MtT测试的人USC的3D培养系统。我们假设人类 USC在3D类器官中保持端粒酶活性和线粒体功能，这大大扩展了 寿命的细胞培养系统，使长期评估晚期ART诱导的MtT。为了验证这个假设， 我们提出以下建议：目标1。评估人USC的干性特性和线粒体功能， 与USC的2D培养相比，长期培养的3D类器官;目的2。测定细胞毒性， 9种抗逆转录病毒药物在3D USC培养中对Pol-γ、线粒体DNA含量和MtT谱的抑制。我们 测试的抗逆转录病毒药物将在3D培养系统中表现出对MtT的影响， 基于体外MtT测定，将其调节至其有效浓度。因此，使用患者来源的 从尿液中提取细胞生成3D培养物的方法为抗逆转录病毒药物的开发提供了一种有前途的工具 个体化用药在抗HIV药物MtT评估中的应用。 抗逆转录病毒疗法的益处常常受到延迟的药物相关毒性的限制。作为 具有 监测 通常 肝细胞 14 细胞 体外 临床 体外 的 药物 评价 相关 第一 细胞 和 更好的T 本项目的潜在目标 预期 根据 干 和

Summary Antiretroviral therapy (ART) has significantly reduced HIV-related morbidity and mortality. However, therapeutic life expectancy for individuals with HIV increased, the long-term safety of ART has gained increasing attention. Thus, it is highly important to the long-term safety of antiretroviral regimens. Human hepatocyte cell lines in 2D culture are most used to evaluate short-term mitochondrial toxicity (MtT) induced by ART. However, these cannot be used in detection of ART-induced chronic MtT because they cannot survive more than days during toxicity testing. In addition, antiviral drug toxicity screening often requires expensive primary such as peripheral blood monocytes in 2D culture systems. More than 90% of drugs that pass through in 2D culture preclinical studies fail to meet the desired efficacy or safety margins required in subsequent trials. Clearly, 2D cultures have contributed to the poor predictive power of MtT screening assays in . Although long-term toxicity tests are often performed in animal models, the high rates of MtT observed in earlier clinical trials suggest that animal toxicology studies may not be suitable for predicting MtT of antiviral intended for human use. Apparently, there are no adequate 2D cell culture or animal models for late MtT for preclinical drug development. Hence, there is an urgent need to develop more toxicologically and clinically predictive in vitro assays to assess compounds for the potential of late MtT. We were the to demonstrate that stem/progenitor cells exist in human urine, i.e., urine-derived stem cells (USC). These can be obtained using simple, non-invasive and low-cost procedures. USC express telomerase activity possess robust proliferative potential. We recently revealed that 3D culture of USC provides a long-term, microenvironmen for cell growth and proliferation, mimicking in vivo conditions, which may have to improve the predictive outcome of preclinical antiviral drug studies. Thus, is to develop 3D culture systems of human USC for ART-induced MtT testing. We hypothesize that human USC maintain telomerase activity and mitochondrial function in 3D organoids, which considerably extends the life of cell culture systems, enabling long-term assessment of late ART-induced MtT . To test this hypothesis, we propose the following: Aim 1. Assess the stemness properties and mitochondrial function of human USC in 3D organoids over long-term culture, compared to 2D culture of USC; Aim 2. Determine the cytotoxicity, inhibition of Pol-γ, mitochondrial DNA content and MtT profiles of 9 antiretroviral drugs in 3D USC cultures. We that the antiretroviral drugs tested will exhibit effects on MtT in the 3D culture systems and be ranked to their effective concentrations based on in vitro MtT assay. Therefore, the use of patients-derived cells from urine to generate 3D culture assay offers a promising tool for antiretroviral drug development personalized medicine in the assessment of MtT of anti-HIV drugs. benefit of ART is often limited by delayed drug-associated toxicity. As has monitor commonly hepatocytes 14 cells vitro clinical vitro the drugs evaluation relevant first cells and better t potential the goal of this project anticipate according stem and